Method of treatment using a soluble human delta protein fragment

ABSTRACT

The present invention relates to nucleotide sequences of vertebrate Delta genes, and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. In a specific embodiment, the vertebrate Delta protein is a human protein. The invention further relates to fragments (and derivatives and analogs thereof) of Delta which comprise one or more domains of the Delta protein, including but not limited to the intracellular domain, extracellular domain, DSL domain, domain amino-terminal to the DSL domain, transmembrane region, or one or more EGF-like repeats of a Delta protein, or any combination of the foregoing. Antibodies to Delta, its derivatives and analogs, are additionally provided. Methods of production of the Delta proteins, derivatives and analogs, e.g., by recombinant means, are also provided. Therapeutic and diagnostic methods and pharmaceutical compositions are provided. In specific examples, isolated Delta genes, from  Xenopus , chick, mouse, and human, are provided.

The present application is a continuation application of applicationSer. No. 09/783,931 filed Feb. 15, 2001, now U.S. Pat. No. 7,118,890 B2,which is a divisional application of application Ser. No. 08/981,392,filed Apr. 7, 1998, now U.S. Pat. No. 6,262,025, which is the nationalstage of International Application No. PCT/US96/11178 filed Jun. 28,1996, which claims the benefit of provisional Application Ser. No.60/000,589 filed Jun. 28, 1995, each of which is incorporated byreference herein in its entirety.

1. INTRODUCTION

The present invention relates to vertebrate Delta genes and theirencoded protein products, as well as derivatives and analogs thereof.Production of vertebrate Delta proteins, derivatives, and antibodies isalso provided. The invention further relates to therapeutic compositionsand methods of diagnosis and therapy.

2. BACKGROUND OF THE INVENTION

Genetic analyses in Drosophila have been extremely useful in dissectingthe complexity of developmental pathways and identifying interactingloci. However, understanding the precise nature of the processes thatunderlie genetic interactions requires a knowledge of the proteinproducts of the genes in question.

The vertebrate central nervous system is an intimate mixture ofdifferent cell types, almost all generated from the same source—theneurogenic epithelium that forms the neural plate and subsequently theneural tube. What are the mechanisms that control neurogenesis in thissheet of cells, directing some to become neurons while others remainnon-neuronal? The answer is virtually unknown for vertebrates, but manyof the cellular interactions and genes controlling cell fate decisionsduring neurogenesis have been well characterized in Drosophila(Campos-Ortega, 1993, J. Neurobiol. 24:1305-1327). Although the grossanatomical context of neurogenesis appears very different in insects andvertebrates, the possibility remains that, at a cellular level, similarevents are occurring via conserved molecular mechanisms. Embryological,genetic and molecular evidence indicates that the early steps ofectodermal differentiation in Drosophila depend on cell interactions(Doe and Goodman, 1985, Dev. Biol. 111:206-219; Technau andCampos-Ortega, 1986, Dev. Biol. 195:445-454; Vässin et al., 1985, J.Neurogenet. 2:291-308; de la Concha et al., 1988, Genetics 118:499-508;Xu et al., 1990, Genes Dev. 4:464-475; Artavanis-Tsakonas, 1988, TrendsGenet. 4:95-100). Mutational analyses reveal a small group ofzygotically-acting genes, the so called neurogenic loci, which affectthe choice of ectodermal cells between epidermal and neural pathways(Poulson, 1937, Proc. Natl. Acad. Sci. 23:133-137; Lehmann et al., 1983,Wilhelm Roux's Arch. Dev. Biol. 192:62-74; Jürgens et al., 1984, WilhelmRoux's Arch. Dev. Biol. 193:283-295; Wieschaus et al., 1984, WilhelmRoux's Arch. Dev. Biol. 193:296-307; Nüsslein-Volhard et al., 1984,Wilhelm Roux's Arch. Dev. Biol. 193:267-282). Null mutations in any oneof the zygotic neurogenic loci—Notch (N), Delta (D1), mastermind (mam),Enhancer of Split (E(spl), neuralized (neu), and big brain (bib)—resultin hypertrophy of the nervous system at the expense of ventral andlateral epidermal structures. This effect is due to the misrouting ofepidermal precursor cells into a neuronal pathway, and implies thatneurogenic gene function is necessary to divert cells within theneurogenic region from a neuronal fate to an epithelial fate.

Neural precursors arise in the Drosophila embryo from a neurogenicepithelium during successive waves of neurogenesis (Campos-Ortega &Hartenstein, 1985, The embryonic development of Drosophila melanogaster(Springer-Verlag, Berlin; N.Y.); Doe, 1992, Development 116:855-863).The pattern of production of these cells is largely determined by theactivity of the proneural and neurogenic genes. Proneural genespredispose clusters of cells to a neural fate (reviewed in Skeath &Carroll, 1994, Faseb J. 8:714-21), but only a subset of cells in acluster become neural precursors. This restriction is due to the actionof the neurogenic genes, which mediate lateral inhibition—a type ofinhibitory cell signaling by which a cell committed to a neural fateforces its neighbors either to remain uncommitted or to enter anon-neural pathway (Artavanis-Tsakonas & Simpson, 1991, Trends Genet.7:403-408; Doe & Goodman, 1985, Dev. Biol. 111:206-219). Mutationsleading to a failure of lateral inhibition cause an overproduction ofneurons—the “neurogenic” phenotype (Lehmann et al., 1981, Roux's Arch.Dev. Biol. 190:226-229; Lehmann et al., Roux's Arch. Dev. Biol.192:62-74). In Drosophila, the inhibitory signal is delivered by atransmembrane protein encoded by the Delta neurogenic gene, which isdisplayed by the nascent neural cells (Heitzler & Simpson, 1991, Cell64:1083-1092). Neighboring cells express a transmembrane receptorprotein, encoded by the neurogenic gene Notch (Fortini &Artavanis-Tsakonas, 1993, Cell 75:1245-1247). Delta has been identifiedas a genetic unit capable of interacting with the Notch locus (Xu etal., 1990, Genes Dev. 4:464-475).

Mutational analyses also reveal that the action of the neurogenic genesis pleiotropic and is not limited solely to embryogenesis. For example,ommatidial, bristle and wing formation, which are known also to dependupon cell interactions, are affected by neurogenic mutations (Morgan etal., 1925, Bibliogr. Genet. 2:1-226; Welshons, 1956, Dros. Inf. Serv.30:157-158; Preiss et al., 1988, EMBO J. 7:3917-3927; Shellenbarger andMohler, 1978, Dev. Biol. 62:432-446; Technau and Campos-Ortega, 1986,Wilhelm Roux's Dev. Biol. 195:445-454; Tomlison and Ready, 1987, Dev.Biol. 120:366-376; Cagan and Ready, 1989, Genes Dev. 3:1099-1112).Neurogenic genes are also required for normal development of themuscles, gut, excretory and reproductive systems of the fly (Muskavitch,1994, Dev. Biol. 166:415-430).

Both Notch and Delta are transmembrane proteins that span the membrane asingle time (Wharton et al., 1985, Cell 43:567-581; Kidd and Young,1986, Mol. Cell. Biol. 6:3094-3108; Vässin, et al., 1987, EMBO J.6:3431-3440; Kopczynski, et al., 1988, Genes Dev. 2:1723-1735) andinclude multiple tandem EGF-like repeats in their extracellular domains(Muskavitch, 1994, Dev. Biol. 166:415-430). The Notch gene encodes a˜300 kd protein (we use “Notch” to denote this protein) with a largeN-terminal extracellular domain that includes 36 epidermal growth factor(EGF)-like tandem repeats followed by three other cysteine-rich repeats,designated Notch/lin-12 repeats (Wharton, et al., 1985, Cell 43:567-581;Kidd and Young, 1986, Mol. Cell. Biol. 6:3094-3108; Yochem, et al.,1988, Nature 335:547-550). Molecular studies have lead to the suggestionthat Notch and Delta constitute biochemically interacting elements of acell communication mechanism involved in early developmental decisions(Fehon et al., 1990, Cell 61:523-534). Homologs are found inCaenorhabditis elegans, where the Notch-related gene lin-12 and theDelta-related gene lag-2 are also responsible for lateral inhibition(Sternberg, 1993, Current Biol. 3:763-765; Henderson et al., 1994,Development 120:2913-2924; Greenwald, 1994, Curr. Opin. Genet. Dev. 204:556-562). In vertebrates, several Notch homologs have also beenidentified (Kopan & Weintraub, 1993, J. Cell Biol. 121:631-641; Lardelliet al., 1994, Mech. Dev. 46:123-136; Lardelli & Lendahl, 1993, Exp. CellRes. 204:364-372; Weinmaster et al., 1991, Development 113:199-205;Weinmaster et al., 1992, Development 116:931-941; Coffman et al., 1990,Science 249:1438-1441; Bierkamp & Campos-Ortega, 1993, Mech. Dev.43:87-100), and they are expressed in many tissues and at many stages ofdevelopment. Loss of Notch-1 leads to somite defects and embryonic deathin mice (Swiatek et al., 1994, Genes Dev. 8:707-719; Conlon et al.,Rossant, J. Development (J. Dev. 121:1533-1545), while constitutivelyactive mutant forms of Notch-1 appear to inhibit cell differentiation inXenopus and in cultured mammalian cells (Coffman et al., 1993, Cell73:659-671; Kopan et al., 1994, Development 120:2385-2396; Nye et al.,1994, Development 120:2421-2430).

The EGF-like motif has been found in a variety of proteins, includingthose involved in the blood clotting cascade (Furie and Furie, 1988,Cell 53: 505-518). In particular, this motif has been found inextracellular proteins such as the blood clotting factors IX and X (Reeset al., 1988, EMBO J. 7:2053-2061; Furie and Furie, 1988, Cell 53:505-518), in other Drosophila genes (Knust et al., 1987 EMBO J. 761-766;Rothberg et al., 1988, Cell 55:1047-1059), and in some cell-surfacereceptor proteins, such as thrombomodulin (Suzuki et al., 1987, EMBO J.6:1891-1897) and LDL receptor (Sudhof et al., 1985, Science228:815-822). A protein binding site has been mapped to the EGF repeatdomain in thrombomodulin and urokinase (Kurosawa et al., 1988, J. Biol.Chem 263:5993-5996; Appella et al., 1987, J. Biol. Chem. 262:4437-4440).

Citation of references hereinabove shall not be construed as anadmission that such references are prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates to nucleotide sequences of vertebrateDelta genes (chick and mouse Delta, and related genes of other species),and amino acid sequences of their encoded proteins, as well asderivatives (e.g., fragments) and analogs thereof. Nucleic acidshybridizable to or complementary to the foregoing nucleotide sequencesare also provided. In a specific embodiment, the Delta protein is amammalian protein, preferably a human protein.

The invention relates to vertebrate Delta derivatives and analogs of theinvention which are functionally active, i.e., they are capable ofdisplaying one or more known functional activities associated with afull-length (wild-type) Delta protein. Such functional activitiesinclude but are not limited to antigenicity [ability to bind (or competewith Delta for binding) to an anti-Delta antibody], immunogenicity(ability to generate antibody which binds to Delta), ability to bind (orcompete with Delta for binding) to Notch or other toporythmic proteinsor fragments thereof (“adhesiveness”), ability to bind (or compete withDelta for binding) to a receptor for Delta. “Toporythmic proteins” asused herein, refers to the protein products of Notch, Delta, Serrate,Enhancer of split, and Deltex, as well as other members of thisinteracting set of genes which may be identified, e.g., by virtue of theability of their gene sequences to hybridize, or their homology toDelta, Serrate, or Notch, or the ability of their genes to displayphenotypic interactions or the ability of their protein products tointeract biochemically.

The invention further relates to fragments (and derivatives and analogsthereof) of a vertebrate Delta that comprise one or more domains of theDelta protein, including but not limited to the intracellular domain,extracellular domain, transmembrane domain, DSL domain, domainamino-terminal to the DSL domain, or one or more EGF-like (homologous)repeats of a Delta protein, or any combination of the foregoing.

Antibodies to a vertebrate Delta, its derivatives and analogs, areadditionally provided.

Methods of production of the vertebrate Delta proteins, derivatives andanalogs, e.g., by recombinant means, are also provided.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on Delta proteins and nucleic acids. Theinvention provides for treatment of disorders of cell fate ordifferentiation by administration of a therapeutic compound of theinvention. Such therapeutic compounds (termed herein “Therapeutics”)include: Delta proteins and analogs and derivatives (includingfragments) thereof; antibodies thereto; nucleic acids encoding the Deltaproteins, analogs, or derivatives; and Delta antisense nucleic acids. Ina preferred embodiment, a Therapeutic of the invention is administeredto treat a cancerous condition, or to prevent progression from apre-neoplastic or non-malignant state into a neoplastic or a malignantstate. In other specific embodiments, a Therapeutic of the invention isadministered to treat a nervous system disorder or to promote tissueregeneration and repair.

In one embodiment, Therapeutics which antagonize, or inhibit, Notchand/or Delta function (hereinafter “Antagonist Therapeutics”) areadministered for therapeutic effect. In another embodiment, Therapeuticswhich promote Notch and/or Delta function (hereinafter “AgonistTherapeutics”) are administered for therapeutic effect.

Disorders of cell fate, in particular hyperproliferative (e.g., cancer)or hypoproliferative disorders, involving aberrant or undesirable levelsof expression or activity or localization of Notch and/or Delta proteincan be diagnosed by detecting such levels, as described more fullyinfra.

In a preferred aspect, a Therapeutic of the invention is a proteinconsisting of at least a fragment (termed herein “adhesive fragment”) ofDelta which mediates binding to a Notch protein or a fragment thereof.

3.1. DEFINITIONS

As used herein, underscoring or italicizing the name of a gene shallindicate the gene, in contrast to its encoded protein product which isindicated by the name of the gene in the absence of any underscoring.For example, “Delta” shall mean the Delta gene, whereas “Delta” shallindicate the protein product of the Delta gene.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A1-1A3-1B1-1B2. FIGS. 1A1-1A3. The DNA sequence of chick Delta(C-Delta-1) (SEQ ID NO:1). FIGS. 1B1-1B2. The DNA sequence of analternatively spliced chick Delta (C-Delta-1) (SEQ ID NO:3).

FIG. 2. The predicted amino acid sequence of chick Delta (C-Delta-1)(SEQ ID NO:2).

FIGS. 3A-3B. Predicted amino acid sequence of C-Delta-1 (SEQ ID NO:2),aligned with that of X-Delta-1 (Xenopus Delta; SEQ ID NO:5) andDrosophila Delta (SEQ ID NO:6) and, indicating the conserved domainstructures: EGF repeats, DSL domain, and transmembrane domain (TM).Conserved amino acids are boxed, and ● denote aligned and non-alignedN-terminal cysteine residues, respectively. Although the intracellulardomains of C-Delta-1 and X-Delta-1 closely resemble each other, theyshow no significant homology to the corresponding part of DrosophilaDelta.

FIG. 4. Alignment of DSL domains from C-Delta-1 (SEQ ID NO:2),Drosophila Delta (SEQ ID NO:6) (Vässin et al., 1987, EMBO J.6:3431-3440; Kopczynski et al., 1988, Genes Dev. 2:1723-1735),Drosophila Serrate (SEQ ID NO:7) (Fleming et al., 1990, Genes Dev.4:2188-2201; Thomas et al., 1991, Development 111:749-761), C-Serrate-1(SEQ ID NO:8) (Myat, Henrique, Ish-Horowicz and Lewis, in preparation),Apx-1 (SEQ ID NO:9) (Mello et al., 1994, Cell 77:95-106) and Lag-2 (SEQID NO:10) (Henderson et al., 1994, Development 120:2913-2924; Tax etal., 1994, Nature 368:150-154), showing the conserved Cysteine spacings,the amino acids that are conserved between presumed ligands forNotch-like proteins in Drosophila and vertebrates, and those that arefurther conserved in C. elegans ligands (boxes).

FIG. 5A-5E. C-Delta-1 and C-Notch-1 expression correlate with onset ofneurogenesis in the one-day (E1) neural plate. Anterior is to the left.Wholemount in situ hybridization specimens are shown in FIG. 5 a-d; 5 eis a section. FIG. 5 a, At stage 7, C-Notch-1 is expressed throughoutmost of the neural plate and part of the underlying presomitic mesoderm.FIG. 5 b, C-Delta-1 at stage 7 is already detectable in the neuralplate, in the future posterior hindbrain, just anterior to the firstsomite (white box). The posterior end of this neural domain is roughlylevel with the anterior margin of a domain of very strong expression inthe underlying presomitic mesoderm (psm). Earlier expression in theneural plate may occur and be masked by expression in the underlyingmesoderm (unpublished results). FIG. 5 c, Higher magnification view ofthe area boxed in 5 b, showing scattered cells in the neural plateexpressing C-Delta-1. FIG. 5 d, At stage 8, C-Delta-1 expression in theneural plate extends posteriorly as the neural plate develops. Thedomain of labelled neural plate cells visible in this photograph(bracketed) continues posteriorly over the presomitic mesoderm. FIG. 5e, Parasagittal section of a stage 8 embryo showing that C-Delta-1 isexpressed in scattered cells of the neural plate (dorsal layer oftissue; bracketed), and broadly in the presomitic mesoderm (ventrallayer). The plane of section is slightly oblique, missing the posteriorpart of the neural plate domain (cf. 5 d).

FIG. 6A-6C. C-Delta-1-expressing cells do not incorporate BrdU. Of 612C-Delta-1⁺ cells, 581 were BrdU⁻ (76 sections; 6 embryos). FIG. 6 a,Diagram showing how phase in the cell cycle is related to apico-basalposition of the nucleus for cells in the neuroepithelium; S-phase nucleilie basally (Fujita, 1963, J. Comp. Neurol. 120:37-42; Biffo et al.,1992, Histochem. Cytochem. 40:535-540). Nuclei are indicated by shading.FIG. 6 b, Section through the neural tube of a stage 9 embryo labelledfor 2 h with BrdU showing C-Delta-i expressing cells (dark on bluebackground) and BrdU-labelled nuclei (pink). Labelled nuclei arepredominantly basal, where DNA synthesis occurs, yet basalC-Delta-1-expressing cells are unlabelled. FIG. 6 c, Section through astage 9 embryo incubated for 4 h: many labelled nuclei have exitedS-phase and have moved towards the lumen, but C-Delta-1-expressing cellsare still basal and not labelled with BrdU.

FIGS. 7A-7B. The DNA sequence of mouse Delta (M-Delta-1) (SEQ ID NO:11).

FIG. 8. The predicted amino acid sequence of the mouse Delta (M-Delta-1)(SEQ ID NO:12).

FIGS. 9A-9B. An alignment of the predicted amino acid sequence of mouseM-Delta-1 (SEQ ID NO:12) with the chick C-Delta-1 (SEQ ID NO:2) whichshows their extensive amino acid sequence identity. Identical aminoacids are boxed. The consensus sequence between the two genes is at thebottom (SEQ ID NO:13).

FIGS. 10A-10B. The DNA sequence of a PCR amplified fragment of humanDelta (H-Delta-1) (SEQ ID NO:14) and the predicted amino acid sequencesusing the three available open reading frames, 2nd line (SEQ IDNO:15-17), 3rd line (SEQ ID NO:18), 4th line (SEQ ID NO:19-22).

FIG. 11. An alignment of human H-Delta-1 (top line) and chick C-Delta-1(bottom line). The predicted amino acid sequence of human Delta (SEQ IDNO:18) is shown in the top line. The sequence of human Delta wasdetermined by “eye”, in which the sequence of the appropriate readingframe was determined by maximizing homology with C-Delta-1(SEQ ID NO:2).No single reading frame shown in FIGS. 10A-10B gave the correct sequencedue to errors in the DNA sequence of FIG. 10 that caused readingframeshifts.

FIGS. 12A1-12A3-12B1-12B6. FIGS. 12A1-12A3 present the contig DNAsequence of human Delta (H-Delta-1) (SEQ ID NO:26) from clone HD118.FIGS. 12B1-12B6 present the nucleotide sequence shown in FIGS. 12A1-12A3(top line, SEQ ID NO:26) and the deduced amino acid sequences using thethree possible open reading frames, second line (SEQ ID NOS:27-42),third line (SEQ ID NOS:43-47), fourth line (SEQ ID NOS:48-64). The aminoacid sequence with the greatest homology to the mouse Delta-1 amino acidsequence is boxed. This boxed amino acid sequence is the predicted aminoacid sequence of human Delta; where the reading frame shifts indicateswhere a sequencing error is present in the sequence. No single readingframe shown in FIGS. 12A1-12A3 gave an uninterrupted amino acid sequencedue to errors in the DNA sequence that caused shifts in the readingframe. X indicates an undetermined amino acid; N indicates anundetermined nucleotide.

FIGS. 13A-13G. An alignment of mouse M-Delta-1 DNA sequence (top line,SEQ ID NO:4) and human H-Delta-1 DNA sequence (second line, SEQ IDNO:26) and their consensus sequence (third line, SEQ ID NO:24).

FIGS. 14A-14B. The composite human Delta (H-Delta-1) amino acid sequence(SEQ ID NOS:65-80, respectively) is presented, representing the boxedamino sequence from FIGS. 12B1-12B6. “>” indicates that the sequencecontinues on the line below. “*” indicates a break in the sequence.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleotide sequences of vertebrateDelta genes, and amino acid sequences of their encoded proteins. Theinvention further relates to fragments and other derivatives, andanalogs, of vertebrate Delta proteins. Nucleic acids encoding suchfragments or derivatives are also within the scope of the invention. Theinvention provides Delta genes and their encoded proteins of manydifferent vertebrate species. The Delta genes of the invention includechick, mouse, and human Delta and related genes (homologs) in othervertebrate species. In specific embodiments, the Delta genes andproteins are from vertebrates, or more particularly, mammals. In apreferred embodiment of the invention, the Delta protein is a humanprotein. Production of the foregoing proteins and derivatives, e.g., byrecombinant methods, is provided.

The invention relates to Delta derivatives and analogs of the inventionwhich are functionally active, i.e., they are capable of displaying oneor more known functional activities associated with a full-length(wild-type) Delta protein. Such functional activities include but arenot limited to antigenicity [ability to bind (or compete with Delta forbinding) to an anti-Delta antibody], immunogenicity (ability to generateantibody which binds to Delta), ability to bind (or compete with Deltafor binding) to Notch or other toporythmic proteins or fragments thereof(“adhesiveness”), ability to bind (or compete with Delta for binding) toa receptor for Delta, ability to affect cell fate differentiation, andtherapeutic activity. “Toporythmic proteins” as used herein, refers tothe protein products of Notch, Delta, Serrate, Enhancer of split, andDeltex, as well as other members of this interacting gene family whichmay be identified, e.g., by virtue of the ability of their genesequences to hybridize, or their homology to Delta, Serrate, or Notch,or the ability of their genes to display phenotypic interactions.

The invention further relates to fragments (and derivatives and analogsthereof) of Delta which comprise one or more domains of the Deltaprotein, including but not limited to the intracellular domain,extracellular domain, DSL domain, region amino-terminal to the DSLdomain, transmembrane domain, membrane-associated region, or one or moreEGF-like (homologous) repeats of a Delta protein, or any combination ofthe foregoing.

Antibodies to vertebrate Delta, its derivatives and analogs, areadditionally provided.

As demonstrated infra, Delta plays a critical role in development andother physiological processes, in particular, as a ligand to Notch,which is involved in cell fate (differentiation) determination. Inparticular, Delta is believed to play a major role in determining cellfates in the central nervous system. The nucleic acid and amino acidsequences and antibodies thereto of the invention can be used for thedetection and quantitation of Delta mRNA and protein of human and otherspecies, to study expression thereof, to produce Delta and fragments andother derivatives and analogs thereof, in the study and manipulation ofdifferentiation and other physiological processes. The present inventionalso relates to therapeutic and diagnostic methods and compositionsbased on Delta proteins and nucleic acids. The invention provides fortreatment of disorders of cell fate or differentiation by administrationof a therapeutic compound of the invention. Such therapeutic compounds(termed herein “Therapeutics”) include: Delta proteins and analogs andderivatives (including fragments) thereof; antibodies thereto; nucleicacids encoding the Delta proteins, analogs, or derivatives; and Deltaantisense nucleic acids. In a preferred embodiment, a Therapeutic of theinvention is administered to treat a cancerous condition, or to preventprogression from a pre-neoplastic or non-malignant state into aneoplastic or a malignant state. In other specific embodiments, aTherapeutic of the invention is administered to treat a nervous systemdisorder or to promote tissue regeneration and repair.

In one embodiment, Therapeutics which antagonize, or inhibit, Notchand/or Delta function (hereinafter “Antagonist Therapeutics”) areadministered for therapeutic effect. In another embodiment, Therapeuticswhich promote Notch and/or Delta function (hereinafter “AgonistTherapeutics”) are administered for therapeutic effect.

Disorders of cell fate, in particular hyperproliferative (e.g., cancer)or hypoproliferative disorders, involving aberrant or undesirable levelsof expression or activity or localization of Notch and/or Delta proteincan be diagnosed by detecting such levels, as described more fullyinfra.

In a preferred aspect, a Therapeutic of the invention is a proteinconsisting of at least a fragment (termed herein “adhesive fragment”) ofDelta which mediates binding to a Notch protein or a fragment thereof.

The invention is illustrated by way of examples infra which disclose,inter alia, the cloning of a chick Delta homolog (Section 6), thecloning of a mouse Delta homolog (Section 7), and the cloning of a humanDelta homolog (Section 8).

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

5.1. Isolation of the Delta Genes

The invention relates to the nucleotide sequences of vertebrate Deltanucleic acids. In specific embodiments, human Delta nucleic acidscomprise the cDNA sequences shown in FIG. 10A-10B (SEQ ID NO:14) or inFIGS. 12A1-12A3 (SEQ ID NO:26), or the coding regions thereof, ornucleic acids encoding a vertebrate Delta protein (e.g., having thesequence of SEQ ID NO:1, 3, 11, 14 or 26). The invention providesnucleic acids consisting of at least 8 nucleotides (i.e., a hybridizableportion) of a vertebrate Delta sequence; in other embodiments, thenucleic acids consist of at least 25 (continuous) nucleotides, 50nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of aDelta sequence, or a full-length Delta coding sequence. The inventionalso relates to nucleic acids hybridizable to or complementary to theforegoing sequences or their complements. In specific aspects, nucleicacids are provided which comprise a sequence complementary to at least10, 25, 50, 100, or 200 nucleotides or the entire coding region of avertebrate Delta gene. In a specific embodiment, a nucleic acid which ishybridizable to a vertebrate (e.g., mammalian) Delta nucleic acid (e.g.,having sequence SEQ ID NO:14 or SEQ ID NO:26, or an at least 10, 25, 50,100, or 200 nucleotide portion thereof), or to a nucleic acid encoding aDelta derivative, under conditions of low stringency is provided. By wayof example and not limitation, procedures using such conditions of lowstringency are as follows (see also Shilo and Weinberg, 1981, Proc.Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA arepretreated for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA,and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried outin the same solution with the following modifications: 0.02% PVP, 0.02%Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextransulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 h at 40° C., and thenwashed for 1.5 h at 55° C. in a solution containing 2× SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and reexposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

In another specific embodiment, a nucleic acid which is hybridizable toa vertebrate (e.g., mammalian) Delta nucleic acid under conditions ofhigh stringency is provided. By way of example and not limitation,procedures using such conditions of high stringency are as follows:Prehybridization of filters containing DNA is carried out for 8 h toovernight at 65° C. in buffer composed of 6× SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. inprehybridization mixture containing 100 μg/ml denatured salmon sperm DNAand 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C. for 1 h in a solution containing 2× SSC, 0.01% PVP, 0.01% Ficoll, and0.01% BSA. This is followed by a wash in 0.1× SSC at 50° C. for 45 minbefore autoradiography. Other conditions of high stringency which may beused are well known in the art.

Nucleic acids encoding fragments and derivatives of vertebrate Deltaproteins (see Section 5.6), and Delta antisense nucleic acids (seeSection 5.11) are additionally provided. As is readily apparent, as usedherein, a “nucleic acid encoding a fragment or portion of a Deltaprotein” shall be construed as referring to a nucleic acid encoding onlythe recited fragment or portion of the Delta protein and not the othercontiguous portions of the Delta protein as a continuous sequence.

Fragments of vertebrate Delta nucleic acids comprising regions ofhomology to other toporythmic proteins are also provided. The DSLregions (regions of homology with Drosophila Serrate and Delta) of Deltaproteins of other species are also provided. Nucleic acids encodingconserved regions between Delta and Serrate, such as those shown inFIGS. 3A-3B and 8 are also provided.

Specific embodiments for the cloning of a vertebrate Delta gene,presented as a particular example but not by way of limitation, follows:

For expression cloning (a technique commonly known in the art), anexpression library is constructed by methods known in the art. Forexample, mRNA (e.g., human) is isolated, cDNA is made and ligated intoan expression vector (e.g., a bacteriophage derivative) such that it iscapable of being expressed by the host cell into which it is thenintroduced. Various screening assays can then be used to select for theexpressed Delta product. In one embodiment, anti-Delta antibodies can beused for selection.

In another preferred aspect, PCR is used to amplify the desired sequencein a genomic or cDNA library, prior to selection. Oligonucleotideprimers representing known Delta sequences (preferably vertebratesequences) can be used as primers in PCR. In a preferred aspect, theoligonucleotide primers represent at least part of the Delta conservedsegments of strong homology between Serrate and Delta. The syntheticoligonucleotides may be utilized as primers to amplify by PCR sequencesfrom a source (RNA or DNA), preferably a cDNA library, of potentialinterest. PCR can be carried out, e.g., by use of a Perkin-Elmer Cetusthermal cycler and Taq polymerase (Gene Amp⁻). The DNA being amplifiedcan include mRNA or cDNA or genomic DNA from any eukaryotic species. Onecan choose to synthesize several different degenerate primers, for usein the PCR reactions. It is also possible to vary the stringency ofhybridization conditions used in priming the PCR reactions, to allow forgreater or lesser degrees of nucleotide sequence similarity between theknown Delta nucleotide sequence and the nucleic acid homolog beingisolated. For cross species hybridization, low stringency conditions arepreferred. For same species hybridization, moderately stringentconditions are preferred. After successful amplification of a segment ofa Delta homolog, that segment may be molecularly cloned and sequenced,and utilized as a probe to isolate a complete cDNA or genomic clone.This, in turn, will permit the determination of the gene's completenucleotide sequence, the analysis of its expression, and the productionof its protein product for functional analysis, as described infra. Inthis fashion, additional genes encoding Delta proteins may beidentified. Such a procedure is presented by way of example in variousexamples sections infra.

The above-methods are not meant to limit the following generaldescription of methods by which clones of Delta may be obtained.

Any vertebrate cell potentially can serve as the nucleic acid source forthe molecular cloning of the Delta gene. The nucleic acid sequencesencoding Delta can be isolated from mammalian, human, porcine, bovine,feline, avian, equine, canine, as well as additional primate sources,etc. For example, we have amplified fragments of the Delta gene inmouse, chicken, and human, by PCR using cDNA libraries with Deltaprimers. The DNA may be obtained by standard procedures known in the artfrom cloned DNA (e.g., a DNA “library”), by chemical synthesis, by cDNAcloning, or by the cloning of genomic DNA, or fragments thereof,purified from the desired cell. (See, for example, Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985,DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I,II.) Clones derived from genomic DNA may contain regulatory and intronDNA regions in addition to coding regions; clones derived from cDNA willcontain only exon sequences. Whatever the source, the gene should bemolecularly cloned into a suitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, if an amount of a portion of a Delta (of any species)gene or its specific RNA, or a fragment thereof, e.g., an extracellulardomain (see Section 5.6), is available and can be purified and labeled,the generated DNA fragments may be screened by nucleic acidhybridization to the labeled probe (Benton, W. and Davis, R., 1977,Science 196:180; Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad.Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology tothe probe will hybridize. It is also possible to identify theappropriate fragment by restriction enzyme digestion(s) and comparisonof fragment sizes with those expected according to a known restrictionmap if such is available. Further selection can be carried out on thebasis of the properties of the gene. Alternatively, the presence of thegene may be detected by assays based on the physical, chemical, orimmunological properties of its expressed product. For example, cDNAclones, or DNA clones which hybrid-select the proper mRNAs, can beselected which produce a protein that, e.g., has similar or identicalelectrophoretic migration, isolectric focusing behavior, proteolyticdigestion maps, binding activity, in vitro aggregation activity(“adhesiveness”) or antigenic properties as known for Delta. If anantibody to Delta is available, the Delta protein may be identified bybinding of labeled antibody to the putatively Delta synthesizing clones,in an ELISA (enzyme-linked immunosorbent assay)-type procedure.

The Delta gene can also be identified by mRNA selection by nucleic acidhybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridization. SuchDNA fragments may represent available, purified Delta DNA of anotherspecies (e.g., Drosophila). Immunoprecipitation analysis or functionalassays (e.g., aggregation ability in vitro; binding to receptor; seeinfra) of the in vitro translation products of the isolated products ofthe isolated mRNAs identifies the mRNA and, therefore, the complementaryDNA fragments that contain the desired sequences. In addition, specificmRNAs may be selected by adsorption of polysomes isolated from cells toimmobilized antibodies specifically directed against Delta protein. Aradiolabelled Delta cDNA can be synthesized using the selected mRNA(from the adsorbed polysomes) as a template. The radiolabelled mRNA orcDNA may then be used as a probe to identify the Delta DNA fragmentsfrom among other genomic DNA fragments.

Alternatives to isolating the Delta genomic DNA include, but are notlimited to, chemically synthesizing the gene sequence itself from aknown sequence or making cDNA to the mRNA which encodes the Deltaprotein. For example, RNA for cDNA cloning of the Delta gene can beisolated from cells which express Delta. Other methods are possible andwithin the scope of the invention.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas PBR322 or pUC plasmid derivatives. The insertion into a cloningvector can, for example, be accomplished by ligating the DNA fragmentinto a cloning vector which has complementary cohesive termini. However,if the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and Delta genemay be modified by homopolymeric tailing. Recombinant molecules can beintroduced into host cells via transformation, transfection, infection,electroporation, etc., so that many copies of the gene sequence aregenerated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionation, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated Delta gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant DNA molecules from thetransformants and, when necessary, retrieving the inserted gene from theisolated recombinant DNA.

The Delta sequences provided by the instant invention include thosenucleotide sequences encoding substantially the same amino acidsequences as found in native vertebrate Delta proteins, and thoseencoded amino acid sequences with functionally equivalent amino acids,all as described in Section 5.6 infra for Delta derivatives.

5.2. Expression of the Delta Genes

The nucleotide sequence coding for a vertebrate Delta protein or afunctionally active fragment or other derivative thereof (see Section5.6), can be inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. The necessarytranscriptional and translational signals can also be supplied by thenative Delta gene and/or its flanking regions. A variety of host-vectorsystems may be utilized to express the protein-coding sequence. Theseinclude but are not limited to mammalian cell systems infected withvirus (e.g., vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophage,DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors varyin their strengths and specificities. Depending on the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements may be used. In a specific embodiment, the adhesiveportion of the Delta gene is expressed. In other specific embodiments,the human Delta gene is expressed, or a sequence encoding a functionallyactive portion of human Delta. In yet another embodiment, a fragment ofDelta comprising the extracellular domain, or other derivative, oranalog of Delta is expressed.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequence encoding a Delta protein or peptidefragment may be regulated by a second nucleic acid sequence so that theDelta protein or peptide is expressed in a host transformed with therecombinant DNA molecule. For example, expression of a Delta protein maybe controlled by any promoter/enhancer element known in the art.Promoters which may be used to control Delta gene expression include,but are not limited to, the SV40 early promoter region (Bernoist andChambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinantbacteria” in Scientific American, 1980, 242:74-94; plant expressionvectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaicvirus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871),and the promoter of the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phosphatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58;alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

Expression vectors containing Delta gene inserts can be identified bythree general approaches: (a) nucleic acid hybridization, (b) presenceor absence of “marker” gene functions, and (c) expression of insertedsequences. In the first approach, the presence of a foreign geneinserted in an expression vector can be detected by nucleic acidhybridization using probes comprising sequences that are homologous toan inserted toporythmic gene. In the second approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain “marker” gene functions (e.g., thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus, etc.) caused by the insertionof foreign genes in the vector. For example, if the Delta gene isinserted within the marker gene sequence of the vector, recombinantscontaining the Delta insert can be identified by the absence of themarker gene function. In the third approach, recombinant expressionvectors can be identified by assaying the foreign gene product expressedby the recombinant. Such assays can be based, for example, on thephysical or functional properties of the Delta gene product in vitroassay systems, e.g., aggregation (binding) with Notch, binding to areceptor, binding with antibody.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered Delta protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, cleavage [e.g., ofsignal sequence]) of proteins. Appropriate cell lines or host systemscan be chosen to ensure the desired modification and processing of theforeign protein expressed. For example, expression in a bacterial systemcan be used to produce an unglycosylated core protein product.Expression in yeast will produce a glycosylated product. Expression inmammalian cells can be used to ensure “native” glycosylation of aheterologous mammalian Delta protein. Furthermore, different vector/hostexpression systems may effect processing reactions such as proteolyticcleavages to different extents.

In other specific embodiments, the Delta protein, fragment, analog, orderivative may be expressed as a fusion, or chimeric protein product(comprising the protein, fragment, analog, or derivative joined via apeptide bond to a heterologous protein sequence (of a differentprotein)). Such a chimeric product can be made by ligating theappropriate nucleic acid sequences encoding the desired amino acidsequences to each other by methods known in the art, in the propercoding frame, and expressing the chimeric product by methods commonlyknown in the art. Alternatively, such a chimeric product may be made byprotein synthetic techniques, e.g., by use of a peptide synthesizer.

Both cDNA and genomic sequences can be cloned and expressed.

5.3. Identification and Purification of the Delta Gene Products

In particular aspects, the invention provides amino acid sequences of avertebrate Delta, preferably a human Delta, and fragments andderivatives thereof which comprise an antigenic determinant (i.e., canbe recognized by an antibody) or which are otherwise functionallyactive, as well as nucleic acid sequences encoding the foregoing.“Functionally active” material as used herein refers to that materialdisplaying one or more known functional activities associated with afull-length (wild-type) Delta protein, e.g., binding to Notch or aportion thereof, binding to any other Delta ligand, antigenicity(binding to an anti-Delta antibody), etc.

In specific embodiments, the invention provides fragments of a Deltaprotein consisting of at least 6 amino acids, 10 amino acids, 25 aminoacids, 50 amino acids, or of at least 75 amino acids. Moleculescomprising such fragments are also provided. In other embodiments, theproteins comprise or consist essentially of an extracellular domain, DSLdomain, epidermal growth factor-like repeat (ELR) domain, one or anycombination of ELRs, transmembrane domain, or intracellular(cytoplasmic) domain, or a portion which binds to Notch, or anycombination of the foregoing, of a vertebrate Delta protein. Fragments,or proteins comprising fragments, lacking some or all of the foregoingregions of a Delta protein are also provided. Nucleic acids encoding theforegoing are provided.

Once a recombinant which expresses the Delta gene sequence isidentified, the gene product can be analyzed. This is achieved by assaysbased on the physical or functional properties of the product, includingradioactive labelling of the product followed by analysis by gelelectrophoresis, immunoassay, etc.

Once the Delta protein is identified, it may be isolated and purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard technique for the purification ofproteins. The functional properties may be evaluated using any suitableassay (see Section 5.7).

Alternatively, once a Delta protein produced by a recombinant isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the chimeric gene contained in therecombinant. As a result, the protein can be synthesized by standardchemical methods known in the art (e.g., see Hunkapiller, M., et al.,1984, Nature 310:105-111).

In a specific embodiment of the present invention, such Delta proteins,whether produced by recombinant DNA techniques or by chemical syntheticmethods, include but are not limited to those containing, as a primaryamino acid sequence, all or part of the amino acid sequencessubstantially as depicted in FIG. 2, 8, 11 or 14A-14B (SEQ ID NOS:2, 12,23 and 65-80), as well as fragments and other derivatives, and analogsthereof.

5.4. Structure of the Delta Genes and Proteins

The structure of the vertebrate Delta genes and proteins can be analyzedby various methods known in the art.

5.4.1. Genetic Analysis

The cloned DNA or cDNA corresponding to the Delta gene can be analyzedby methods including but not limited to Southern hybridization(Southern, E.M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization(see e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:4094-4098), restriction endonuclease mapping (Maniatis, T., 1982,Molecular Cloning, A Laboratory, Cold Spring Harbor, N.Y.), and DNAsequence analysis. Polymerase chain reaction (PCR; U.S. Pat. Nos.4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc.Natl. Acad. Sci. U.S.A. 85:7652-7656; Ochman et al., 1988, Genetics120:621-623; Loh et al., 1989, Science 243:217-220) followed by Southernhybridization with a Delta-specific probe can allow the detection of theDelta gene in DNA from various cell types. Methods of amplificationother than PCR are commonly known and can also be employed. In oneembodiment, Southern hybridization can be used to determine the geneticlinkage of Delta. Northern hybridization analysis can be used todetermine the expression of the Delta gene. Various cell types, atvarious states of development or activity can be tested for Deltaexpression. Examples of such techniques and their results are describedin Section 6, infra. The stringency of the hybridization conditions forboth Southern and Northern hybridization can be manipulated to ensuredetection of nucleic acids with the desired degree of relatedness to thespecific Delta probe used.

Restriction endonuclease mapping can be used to roughly determine thegenetic structure of the Delta gene. Restriction maps derived byrestriction endonuclease cleavage can be confirmed by DNA sequenceanalysis.

DNA sequence analysis can be performed by any 25 techniques known in theart, including but not limited to the method of Maxam and Gilbert (1980,Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., etal., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNApolymerase (Tabor and Richardson, U.S. Pat. No. 4,795,699), or use of anautomated DNA sequenator (e.g., Applied Biosystems, Foster City,Calif.).

5.4.2. Protein Analysis

The amino acid sequence of the Delta protein can be derived by deductionfrom the DNA sequence, or alternatively, by direct sequencing of theprotein, e.g., with an automated amino acid sequencer. The amino acidsequence of a representative Delta protein comprises the sequencesubstantially as depicted in FIG. 2, and detailed in Section 6, infra,with the representative mature protein that shown by amino acid numbers1-728.

The Delta protein sequence can be further characterized by ahydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:3824). A hydrophilicity profile can be used to identifythe hydrophobic and hydrophilic regions of the Delta protein and thecorresponding regions of the gene sequence which encode such regions.Hydrophilic regions are more likely to be immunogenic.

Secondary, structural analysis (Chou, P. and Fasman, G., 1974,Biochemistry 13:222) can also be done, to identify regions of Delta thatassume specific secondary structures.

Manipulation, translation, and secondary structure prediction, as wellas open reading frame prediction and plotting, can also be accomplishedusing computer software programs available in the art.

Other methods of structural analysis can also be employed. These includebut are not limited to X-ray crystallography (Engstom, A., 1974,Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick, R. andZoller, M. (eds.), 1986, Computer Graphics and Molecular Modeling, inCurrent Communications in Molecular Biology, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

5.5. Generation of Antibodies to Delta Proteins and Derivatives Thereof

According to the invention, a vertebrate Delta protein, its fragments orother derivatives, or analogs thereof, may be used as an immunogen togenerate antibodies which recognize such an immunogen. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library. In a specificembodiment, antibodies to human Delta are produced. In anotherembodiment, antibodies to the extracellular domain of Delta areproduced. In another embodiment, antibodies to the intracellular domainof Delta are produced.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a Delta protein or derivative or analog. In aparticular embodiment, rabbit polyclonal antibodies to an epitope of theDelta protein encoded by a sequence depicted in FIG. 1A1-1A3, 1B1-1B2,7A-7B or 11, or a subsequence thereof, can be obtained. For theproduction of antibody, various host animals can be immunized byinjection with the native Delta protein, or a synthetic version, orderivative (e.g., fragment) thereof, including but not limited torabbits, mice, rats, etc. Various adjuvants may be used to increase theimmunological response, depending on the host species, and including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette-Guerin) and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a Delta proteinsequence or analog thereof, any technique which provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. For example, the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology (PCT/US90/02545).According to the invention, human antibodies may be used and can beobtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBVvirus in vitro (Cole et al., 1985, in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, pp. 77-96). In fact, according to the invention,techniques developed for the production of “chimeric antibodies”(Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing the genes from a mouse antibody moleculespecific for Delta together with genes from a human antibody molecule ofappropriate biological activity can be used; such antibodies are withinthe scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce Delta-specific single chain antibodies. An additional embodimentof the invention utilizes the techniques described for the constructionof Fab expression libraries (Huse et al., 1989, Science 246:1275-1281)to allow rapid and easy identification of monoclonal Fab fragments withthe desired specificity for Delta proteins, derivatives, or analogs.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the (ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, andthe Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize a specific domain of a vertebrate Delta protein, one mayassay generated hybridomas for a product which binds to a Delta fragmentcontaining such domain. For selection of an antibody immunospecific tohuman Delta, one can select on the basis of positive binding to humanDelta and a lack of binding to Drosophila Delta.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the protein sequences ofthe invention (e.g., see Section 5.7, infra), e.g., for imaging theseproteins, measuring levels thereof in appropriate physiological samples,in diagnostic methods, etc.

Antibodies specific to a domain of a Delta protein are also provided. Ina specific embodiment, antibodies which bind to a Notch-binding fragmentof Delta are provided.

In another embodiment of the invention (see infra), anti-Deltaantibodies and fragments thereof containing the binding domain areTherapeutics.

5.6. Delta Proteins, Derivatives and Analogs

The invention further relates to vertebrate (e.g., mammalian) Deltaproteins, and derivatives (including but not limited to fragments) andanalogs of vertebrate Delta proteins. Nucleic acids encoding Deltaprotein derivatives and protein analogs are also provided. In oneembodiment, the Delta proteins are encoded by the Delta nucleic acidsdescribed in Section 5.1 supra. In particular aspects, the proteins,derivatives, or analogs are of mouse, chicken, rat, pig, cow, dog,monkey, or human Delta proteins. In a specific embodiment, a mature,full-length vertebrate Delta protein is provided. In one embodiment, avertebrate Delta protein lacking only the signal sequence (approximatelythe first 17 amino-terminal amino acids) is provided.

The production and use of derivatives and analogs related to Delta arewithin the scope of the present invention. In a specific embodiment, thederivative or analog is functionally active, i.e., capable of exhibitingone or more functional activities associated with a full-length,wild-type Delta protein. As one example, such derivatives or analogswhich have the desired immunogenicity or antigenicity can be used, forexample, in immunoassays, for immunization, for inhibition of Deltaactivity, etc. Such molecules which retain, or alternatively inhibit, adesired Delta property, e.g., binding to Notch or other toporythmicproteins, binding to a cell-surface receptor, can be used as inducers,or inhibitors, respectively, of such property and its physiologicalcorrelates. A specific embodiment relates to a Delta fragment that canbe bound by an anti-Delta antibody but cannot bind to a Notch protein orother toporythmic protein. Derivatives or analogs of Delta can be testedfor the desired activity by procedures known in the art, including butnot limited to the assays described in Section 5.7.

In particular, Delta derivatives can be made by altering Delta sequencesby substitutions, additions or deletions that provide for functionallyequivalent molecules. Due to the degeneracy of nucleotide codingsequences, other DNA sequences which encode substantially the same aminoacid sequence as a Delta gene may be used in the practice of the presentinvention. These include but are not limited to nucleotide sequencescomprising all or portions of Delta genes which are altered by thesubstitution of different codons that encode a functionally equivalentamino acid residue within the sequence, thus producing a silent change.Likewise, the Delta derivatives of the invention include, but are notlimited to, those containing, as a primary amino acid sequence, all orpart of the amino acid sequence of a Delta protein including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. For example, one or more amino acid residues within the sequencecan be substituted by another amino acid of a similar polarity whichacts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

In a specific embodiment of the invention, proteins consisting of orcomprising a fragment of a vertebrate Delta protein consisting of atleast 10 (continuous) amino acids of the Delta protein is provided. Inother embodiments, the fragment consists of at least 20 or 50 aminoacids of the Delta protein. In specific embodiments, such fragments arenot larger than 35, 100 or 200 amino acids. Derivatives or analogs ofDelta include but are not limited to those peptides which aresubstantially homologous to a vertebrate Delta protein or fragmentsthereof (e.g., at least 30%, 50%, 70%, or 90% identity over an aminoacid sequence of identical size—e.g., comprising a domain) or whoseencoding nucleic acid is capable of hybridizing to a coding Deltasequence.

The Delta derivatives and analogs of the invention can be produced byvarious methods known in the art. The manipulations which result intheir production can occur at the gene or protein level. For example,the cloned Delta gene sequence can be modified by any of numerousstrategies known in the art (Maniatis, T., 1990, Molecular Cloning, ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). The sequence can be cleaved at appropriate sites withrestriction endonuclease(s), followed by further enzymatic modificationif desired, isolated, and ligated in vitro. In the production of thegene encoding a derivative or analog of Delta, care should be taken toensure that the modified gene remains within the same translationalreading frame as Delta, uninterrupted by translational stop signals, inthe gene region where the desired Delta activity is encoded.

Additionally, the Delta-encoding nucleic acid sequence can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J.Biol. Chem 253:6551), use of TABS linkers (Pharmacia), etc. PCR primerscontaining sequence changes can be used in PCR to introduce such changesinto the amplified fragments.

Manipulations of the Delta sequence may also be made at the proteinlevel. Included within the scope of the invention are Delta proteinfragments or other derivatives or analogs which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

In addition, analogs and derivatives of Delta can be chemicallysynthesized. For example, a peptide corresponding to a portion of aDelta protein which comprises the desired domain (see Section 5.6.1), orwhich mediates the desired aggregation activity in vitro, or binding toa receptor, can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the Deltasequence. Non-classical amino acids include but are not limited to theD-isomers of the common amino acids, α-amino isobutyric acid,4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteicacid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, designer amino acids such as β-methyl amino acids, Cα-methylamino acids, and Nα-methyl amino acids.

In a specific embodiment, the Delta derivative is a chimeric, or fusion,protein comprising a vertebrate Delta protein or fragment thereof(preferably consisting of at least a domain or motif of the Deltaprotein, or at least 10 amino acids of the Delta protein) joined at itsamino- or carboxy-terminus via a peptide bond to an amino acid sequenceof a different protein. In one embodiment, such a chimeric protein isproduced by recombinant expression of a nucleic acid encoding theprotein (comprising a Delta-coding sequence joined in-frame to a codingsequence for a different protein). Such a chimeric product can be madeby ligating the appropriate nucleic acid sequences encoding the desiredamino acid sequences to each other by methods known in the art, in theproper coding frame, and expressing the chimeric product by methodscommonly known in the art. Alternatively, such a chimeric product may bemade by protein synthetic techniques, e.g., by use of a peptidesynthesizer. In a specific embodiment, a chimeric nucleic acid encodinga mature Delta protein with a heterologous signal sequence is expressedsuch that the chimeric protein is expressed and processed by the cell tothe mature Delta protein. As another example, and not by way oflimitation, a recombinant molecule can be constructed according to theinvention, comprising coding portions of both Delta and anothertoporythmic gene, e.g., Serrate. The encoded protein of such arecombinant molecule could exhibit properties associated with bothSerrate and Delta and portray a novel profile of biological activities,including agonists as well as antagonists. The primary sequence of Deltaand Serrate may also be used to predict tertiary structure of themolecules using computer simulation (Hopp and Woods, 1981, Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828); Delta/Serrate chimeric recombinantgenes could be designed in light of correlations between tertiarystructure and biological function. Likewise, chimeric genes comprisingportions of Delta fused to any heterologous protein-encoding sequencesmay be constructed. A specific embodiment relates to a chimeric proteincomprising a fragment of Delta of at least six amino acids.

In another specific embodiment, the Delta derivative is a fragment ofvertebrate Delta comprising a region of homology with anothertoporythmic protein. As used herein, a region of a first protein shallbe considered “homologous” to a second protein when the amino acidsequence of the region is at least 30% identical or at least 75% eitheridentical or involving conservative changes, when compared to anysequence in the second protein of an equal number of amino acids as thenumber contained in the region. For example, such a Delta fragment cancomprise one or more regions homologous to Serrate, including but notlimited to the DSL domain or a portion thereof.

Other specific embodiments of derivatives and analogs are described inthe subsections below and examples sections infra.

5.6.1. Derivatives of Delta Containing One or More Domains of theProtein

In a specific embodiment, the invention relates to vertebrate Deltaderivatives and analogs, in particular Delta fragments and derivativesof such fragments, that comprise, or alternatively consist of, one ormore domains of the Delta protein, including but not limited to theextracellular domain, signal sequence, region amino-terminal to the DSLdomain, DSL domain, ELR domain, transmembrane domain, intracellulardomain, and one or more of the EGF-like repeats (ELR) of the Deltaprotein (e.g., ELRs 1-9), or any combination of the foregoing. Inparticular examples relating to the chick and mouse Delta proteins, suchdomains are identified in Examples Section 6 and 7, respectively, and inFIGS. 3a-3B and 9A-9B. Thus, by way of example is provided, a moleculecomprising an extracellular domain (approximately amino acids 1-545),signal sequence (approximately amino acids 1-17), region amino-terminalto the DSL domain (approximately amino acids 1-178), the DSL domain(approximately amino acids 179-223), EGF1 (approximately amino acids229-260), EGF2 (approximately amino acids 261-292), EGF3 (approximatelyamino acids 293-332), EGF4 (approximately amino acids 333-370), EGF5(approximately amino acids 371-409), EGF6 (approximately amino acids410-447), EGF7 (approximately amino acids 448-485), EGF8 (approximatelyamino acids 486-523), transmembrane domain, and intracellular(cytoplasmic) domain (approximately amino acids 555-728) of a vertebrateDelta.

In a specific embodiment, the molecules comprising specific fragments ofvertebrate Delta are those comprising fragments in the respective Deltaprotein most homologous to specific fragments of the Drosophila or chickDelta protein. In particular embodiments, such a molecule comprises orconsists of the amino acid sequences of SEQ ID NO:2 or 23.Alternatively, a fragment comprising a domain of a Delta homolog can beidentified by protein analysis methods as described in Section 5.3.2.

5.6.2. Derivatives of Delta that Medicate Binding to Toporythmic ProteinDomains

The invention also provides for vertebrate Delta fragments, and analogsor derivatives of such fragments, which mediate binding to toporythmicproteins (and thus are termed herein “adhesive”), and nucleic acidsequences encoding the foregoing.

In a particular embodiment, the adhesive fragment of a Delta proteincomprises the DSL domain, or a portion thereof. Subfragments within theDSL domain that mediate binding to Notch can be identified by analysisof constructs expressing deletion mutants.

The ability to bind to a toporythmic protein (preferably Notch) can bedemonstrated by in vitro aggregation assays with cells expressing such atoporythmic protein as well as cells expressing Delta or a Deltaderivative (See Section 5.7). That is, the ability of a Delta fragmentto bind to a Notch protein can be demonstrated by detecting the abilityof the Delta fragment, when expressed on the surface of a first cell, tobind to a Notch protein expressed on the surface of a second cell.

The nucleic acid sequences encoding toporythmic proteins or adhesivedomains thereof, for use in such assays, can be isolated from human,porcine, bovine, feline, avian, equine, canine, or insect, as well asprimate sources and any other species in which homologs of knowntoporythmic genes can be identified.

5.7. Assays of Delta Proteins, Derivatives and Analogs

The functional activity of vertebrate Delta proteins, derivatives andanalogs can be assayed by various methods.

For example, in one embodiment, where one is assaying for the ability tobind or compete with wild-type Delta for binding to anti-Delta antibody,various immunoassays known in the art can be used, including but notlimited to competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immunodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labelled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where one is assaying for the ability to mediatebinding to a toporythmic protein, e.g., Notch, one can carry out an invitro aggregation assay (see Fehon et al., 1990, Cell 61:523-534; Rebayet al., 1991, Cell 67:687-699).

In another embodiment, where a receptor for Delta is identified,receptor binding can be assayed, e.g., by means well-known in the art.In another embodiment, physiological correlates of Delta binding tocells expressing a Delta receptor (signal transduction) can be assayed.

In another embodiment, in insect or other model systems, genetic studiescan be done to study the phenotypic effect of a Delta mutant that is aderivative or analog of wild-type Delta.

Other methods will be known to the skilled artisan and are within thescope of the invention.

5.8. Therapeutic Uses

The invention provides for treatment of disorders of cell fate ordifferentiation by administration of a therapeutic compound of theinvention. Such therapeutic compounds (termed herein “Therapeutics”)include: Delta proteins and analogs and derivatives (includingfragments) thereof (e.g., as described hereinabove); antibodies thereto(as described hereinabove); nucleic acids encoding the Delta proteins,analogs, or derivatives (e.g., as described hereinabove); and Deltaantisense nucleic acids. As stated supra, the Antagonist Therapeutics ofthe invention are those Therapeutics which antagonize, or inhibit, aDelta function and/or Notch function (since Delta is a Notch ligand).Such Antagonist Therapeutics are most preferably identified by use ofknown convenient in vitro assays, e.g., based on their ability toinhibit binding of Delta to another protein (e.g., a Notch protein), orinhibit any known Notch or Delta function as preferably assayed in vitroor in cell culture, although genetic assays (e.g., in Drosophila) mayalso be employed. In a preferred embodiment, the Antagonist Therapeuticis a protein or derivative thereof comprising a functionally activefragment such as a fragment of Delta which mediates binding to Notch, oran antibody thereto. In other specific embodiments, such an AntagonistTherapeutic is a nucleic acid capable of expressing a moleculecomprising a fragment of Delta which binds to Notch, or a Deltaantisense nucleic acid (see Section 5.11 herein). It should be notedthat preferably, suitable in vitro or in vivo assays, as describedinfra, should be utilized to determine the effect of a specificTherapeutic and whether its administration is indicated for treatment ofthe affected tissue, since the developmental history of the tissue maydetermine whether an Antagonist or Agonist Therapeutic is desired.

In addition, the mode of administration, e.g., whether administered insoluble form or administered via its encoding nucleic acid forintracellular recombinant expression, of the Delta protein or derivativecan affect whether it acts as an agonist or antagonist.

In another embodiment of the invention, a nucleic acid containing aportion of a Delta gene is used, as an Antagonist Therapeutic, topromote Delta inactivation by homologous recombination (Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra etal., 1989, Nature 342:435-438).

The Agonist Therapeutics of the invention, as described supra, promoteDelta function. Such Agonist Therapeutics include but are not limited toproteins and derivatives comprising the portions of Notch that mediatebinding to Delta, and nucleic acids encoding the foregoing (which can beadministered to express their encoded products in vivo).

Further descriptions and sources of Therapeutics of the inventions arefound in Sections 5.1 through 5.7 herein.

Molecules which retain, or alternatively inhibit, a desired Deltaproperty, e.g., binding to Notch, binding to an intracellular ligand,can be used therapeutically as inducers, or inhibitors, respectively, ofsuch property and its physiological correlates. In a specificembodiment, a peptide (e.g., in the range of 6-50 or 15-25 amino acids;and particularly of about 10, 15, 20 or 25 amino acids) containing thesequence of a portion of Delta which binds to Notch is used toantagonize Notch function. In a specific embodiment, such an AntagonistTherapeutic is used to treat or prevent human or other malignanciesassociated with increased Notch expression (e.g., cervical cancer, coloncancer, breast cancer, squamous adenocarcimas (see infra)). Derivativesor analogs of Delta can be tested for the desired activity by proceduresknown in the art, including but not limited to the assays described inthe examples infra. For example, molecules comprising Delta fragmentswhich bind to Notch EGF-repeats (ELR) 11 and 12 and which are smallerthan a DSL domain, can be obtained and selected by expressing deletionmutants and assaying for binding of the expressed product to Notch byany of the several methods (e.g., in vitro cell aggregation assays,interaction trap system), some of which are described in the ExamplesSections infra. In one specific embodiment, peptide libraries can bescreened to select a peptide with the desired activity; such screeningcan be carried out by assaying, e.g., for binding to Notch or a moleculecontaining the Notch ELR 11 and 12 repeats.

Other Therapeutics include molecules that bind to a vertebrate Deltaprotein. Thus, the invention also provides a method for identifying suchmolecules. Such molecules can be identified by a method comprisingcontacting a plurality of molecules (e.g., in a peptide library, orcombinatorial chemical library) with the Delta protein under conditionsconducive to binding, and recovering any molecules that bind to theDelta protein.

The Agonist and Antagonist Therapeutics of the invention havetherapeutic utility for disorders of cell fate. The Agonist Therapeuticsare administered therapeutically (including prophylactically): (1) indiseases or disorders involving an absence or decreased (relative tonormal, or desired) levels of Notch or Delta function, for example, inpatients where Notch or Delta protein is lacking, genetically defective,biologically inactive or underactive, or underexpressed; and (2) indiseases or disorders wherein in vitro (or in vivo) assays (see infra)indicate the utility of Delta agonist administration. The absence ordecreased levels in Notch or Delta function can be readily detected,e.g., by obtaining a patient tissue sample (e.g., from biopsy tissue)and assaying it in vitro for protein levels, structure and/or activityof the expressed Notch or Delta protein. Many methods standard in theart can be thus employed, including but not limited to immunoassays todetect and/or visualize Notch or Delta protein (e.g., Western blot,immunoprecipitation followed by sodium dodecyl sulfate polyacrylamidegel electrophoresis, immunocytochemistry, etc.) and/or hybridizationassays to detect Notch or Delta expression by detecting and/orvisualizing respectively Notch or Delta mRNA (e.g., Northern assays, dotblots, in situ hybridization, etc.)

In vitro assays which can be used to determine whether administration ofa specific Agonist Therapeutic or Antagonist Therapeutic is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered aTherapeutic, and the effect of such Therapeutic upon the tissue sampleis observed. In one embodiment, where the patient has a malignancy, asample of cells from such malignancy is plated out or grown in culture,and the cells are then exposed to a Therapeutic. A Therapeutic whichinhibits survival or growth of the malignant cells (e.g., by promotingterminal differentiation) is selected for therapeutic use in vivo. Manyassays standard in the art can be used to assess such survival and/orgrowth; for example, cell proliferation can be assayed by measuring³H-thymidine incorporation, by direct cell count, by detecting changesin transcriptional activity of known genes such as proto-oncogenes(e.g., fos, myc) or cell cycle markers; cell viability can be assessedby trypan blue staining, differentiation can be assessed visually basedon changes in morphology, etc. In a specific aspect, the malignant cellcultures are separately exposed to (1) an Agonist Therapeutic, and (2)an Antagonist Therapeutic; the result of the assay can indicate whichtype of Therapeutic has therapeutic efficacy.

In another embodiment, a Therapeutic is indicated for use which exhibitsthe desired effect, inhibition or promotion of cell growth, upon apatient cell sample from tissue having or suspected of having a hyper-or hypoproliferative disorder, respectively. Such hyper- orhypoproliferative disorders include but are not limited to thosedescribed in Sections 5.8.1 through 5.8.3 infra.

In another specific embodiment, a Therapeutic is indicated for use intreating nerve injury or a nervous system degenerative disorder (seeSection 5.8.2) which exhibits in vitro promotion of nerveregeneration/neurite extension from nerve cells of the affected patienttype.

In addition, administration of an Antagonist Therapeutic of theinvention is also indicated in diseases or disorders determined or knownto involve a Notch or Delta dominant activated phenotype (“gain offunction” mutations.) Administration of an Agonist Therapeutic isindicated in diseases or disorders determined or known to involve aNotch or Delta dominant negative phenotype (“loss of function”mutations). The functions of various structural domains of the Notchprotein have been investigated in vivo, by ectopically expressing aseries of Drosophila Notch deletion mutants under the hsp7o heat-shockpromoter, as well as eye-specific promoters (see Rebay et al., 1993,Cell 74:319-329). Two classes of dominant phenotypes were observed, onesuggestive of Notch loss-of function mutations and the other of Notchgain-of-function mutations. Dominant “activated” phenotypes resultedfrom overexpression of a protein lacking most extracellular sequences,while dominant “negative” phenotypes resulted from overexpression of aprotein lacking most intracellular sequences. The results indicated thatNotch functions as a receptor whose extracellular domain mediatesligand-binding, resulting in the transmission of developmental signalsby the cytoplasmic domain.

In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if a Therapeutic has a desired effect upon such cell types.

In another embodiment, cells of a patient tissue sample suspected ofbeing pre-neoplastic are similarly plated out or grown in vitro, andexposed to a Therapeutic. The Therapeutic which results in a cellphenotype that is more normal (i.e., less representative of apre-neoplastic state, neoplastic state, malignant state, or transformedphenotype) is selected for therapeutic use. Many assays standard in theart can be used to assess whether a pre-neoplastic state, neoplasticstate, or a transformed or malignant phenotype, is present. For example,characteristics associated with a transformed phenotype (a set of invitro characteristics associated with a tumorigenic ability in vivo)include a more rounded cell morphology, looser substratum attachment,loss of contact inhibition, loss of anchorage dependence, release ofproteases such as plasminogen activator, increased sugar transport,decreased serum requirement, expression of fetal antigens, disappearanceof the 250,000 dalton surface protein, etc. (see Luria et al., 1978,General Virology, 3d Ed., John Wiley & Sons, New York pp. 436-446).

In other specific embodiments, the in vitro assays described supra canbe carried out using a cell line, rather than a cell sample derived fromthe specific patient to be treated, in which the cell line is derivedfrom or displays characteristic(s) associated with the malignant,neoplastic or pre-neoplastic disorder desired to be treated orprevented, or is derived from the neural or other cell type upon whichan effect is desired, according to the present invention.

The Antagonist Therapeutics are administered therapeutically (includingprophylactically): (1) in diseases or disorders involving increased(relative to normal, or desired) levels of Notch or Delta function, forexample, where the Notch or Delta protein is overexpressed oroveractive; and (2) in diseases or disorders wherein in vitro (or invivo) assays indicate the utility of Delta antagonist administration.The increased levels of Notch or Delta function can be readily detectedby methods such as those described above, by quantifying protein and/orRNA. In vitro assays with cells of patient tissue sample or theappropriate cell line or cell type, to determine therapeutic utility,can be carried out as described above.

5.8.1. Malignancies

Malignant and pre-neoplastic conditions which can be tested as describedsupra for efficacy of intervention with Antagonist or AgonistTherapeutics, and which can be treated upon thus observing an indicationof therapeutic utility, include but are not limited to those describedbelow in Sections 5.8.1 and 5.9.1.

Malignancies and related disorders, cells of which type can be tested invitro (and/or in vivo), and upon observing the appropriate assay result,treated according to the present invention, include but are not limitedto those listed in Table 1 (for a review of such disorders, see Fishmanet al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia):

TABLE 1 MALIGNANCIES AND RELATED DISORDERS Leukemia   acute leukemia    acute lymphocytic leukemia     acute myelocytic leukemia      myeloblastic       promyelocytic       myelomonocytic      monocytic       erythroleukemia   chronic leukemia     chronicmyelocytic (granulocytic) leukemia     chronic lymphocytic leukemiaPolycythemia vera Lymphoma   Hodgkin's disease   non-Hodgkin's diseaseMultiple myeloma Waldenström's macroglobulinemia Heavy chain diseaseSolid tumors   sarcomas and carcinomas     fibrosarcoma     myxosarcoma    liposarcoma     chondrosarcoma     osteogenic sarcoma     chordoma    angiosarcoma     endotheliosarcoma     lymphangiosarcoma    lymphangioendotheliosarcoma     synovioma     mesothelioma    Ewing's tumor     leiomyosarcoma     rhabdomyosarcoma     coloncarcinoma     pancreatic cancer     breast cancer     ovarian cancer    prostate cancer     squamous cell carcinoma     basal cell carcinoma    adenocarcinoma     sweat gland carcinoma     sebaceous glandcarcinoma     papillary carcinoma     papillary adenocarcinomas    cystadenocarcinoma     medullary carcinoma     bronchogeniccarcinoma     renal cell carcinoma     hepatoma     bile duct carcinoma    choriocarcinoma     seminoma     embryonal carcinoma     Wilms'tumor     cervical cancer     testicular tumor     lung carcinoma    small cell lung carcinoma     bladder carcinoma     epithelialcarcinoma     glioma     astrocytoma     medulloblastoma    craniopharyngioma     ependymoma     pinealoma     hemangioblastoma    acoustic neuroma     oligodendroglioma     menangioma     melanoma    neuroblastoma     retinoblastoma

In specific embodiments, malignancy or dysproliferative changes (such asmetaplasias and dysplasias) are treated or prevented in epithelialtissues such as those in the cervix, esophagus, and lung.

Malignancies of the colon and cervix exhibit increased expression ofhuman Notch relative to such non-malignant tissue (see PCT Publicationno. WO 94/07474 published Apr. 14, 1994, incorporated by referenceherein in its entirety). Thus, in specific embodiments, malignancies orpremalignant changes of the colon or cervix are treated or prevented byadministering an effective amount of an Antagonist Therapeutic, e.g., aDelta derivative, that antagonizes Notch function. The presence ofincreased Notch expression in colon, and cervical cancer suggests thatmany more cancerous and hyperproliferative conditions exhibitupregulated Notch. Thus, in specific embodiments, various cancers, e.g.,breast cancer, squamous adenocarcinoma, seminoma, melanoma, and lungcancer, and premalignant changes therein, as well as otherhyperproliferative disorders, can be treated or prevented byadministration of an Antagonist Therapeutic that antagonizes Notchfunction.

5.8.2. Nervous System Disorders

Nervous system disorders, involving cell types which can be tested asdescribed supra for efficacy of intervention with Antagonist or AgonistTherapeutics, and which can be treated upon thus observing an indicationof therapeutic utility, include but are not limited to nervous systeminjuries, and diseases or disorders which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated in a patient(including human and non-human mammalian patients) according to theinvention include but are not limited to the following lesions of eitherthe central (including spinal cord, brain) or peripheral nervoussystems:

-   -   (i) traumatic lesions, including lesions caused by physical        injury or associated with surgery, for example, lesions which        sever a portion of the nervous system, or compression injuries;    -   (ii) ischemic lesions, in which a lack of oxygen in a portion of        the nervous system results in neuronal injury or death,        including cerebral infarction or ischemia, or spinal cord        infarction or ischemia;    -   (iii) malignant lesions, in which a portion of the nervous        system is destroyed or injured by malignant tissue which is        either a nervous system associated malignancy or a malignancy        derived from non-nervous system tissue;    -   (iv) infectious lesions, in which a portion of the nervous        system is destroyed or injured as a result of infection, for        example, by an abscess or associated with infection by human        immunodeficiency virus, herpes zoster, or herpes simplex virus        or with Lyme disease, tuberculosis, syphilis;    -   (v) degenerative lesions, in which a portion of the nervous        system is destroyed or injured as a result of a degenerative        process including but not limited to degeneration associated        with Parkinson's disease, Alzheimer's disease, Huntington's        chorea, or amyotrophic lateral sclerosis;    -   (vi) lesions associated with nutritional diseases or disorders,        in which a portion of the nervous system is destroyed or injured        by a nutritional disorder or disorder of metabolism including        but not limited to, vitamin B12 deficiency, folic acid        deficiency, Wernicke disease, tobacco-alcohol amblyopia,        Marchiafava-Bignami disease (primary degeneration of the corpus        callosum), and alcoholic cerebellar degeneration;    -   (vii) neurological lesions associated with systemic diseases        including but not limited to diabetes (diabetic neuropathy,        Bell's palsy), systemic lupus erythematosus, carcinoma, or        sarcoidosis;    -   (viii) lesions caused by toxic substances including alcohol,        lead, or particular neurotoxins; and    -   (ix) demyelinated lesions in which a portion of the nervous        system is destroyed or injured by a demyelinating disease        including but not limited to multiple sclerosis, human        immunodeficiency virus-associated myelopathy, transverse        myelopathy or various etiologies, progressive multifocal        leukoencephalopathy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatmentof a nervous system disorder may be selected by testing for biologicalactivity in promoting the survival or differentiation of neurons (seealso Section 5.8). For example, and not by way of limitation,Therapeutics which elicit any of the following effects may be usefulaccording to the invention:

-   -   (i) increased survival time of neurons in culture;    -   (ii) increased sprouting of neurons in culture or in vivo;    -   (iii) increased production of a neuron-associated molecule in        culture or in vivo, e.g., choline acetyltransferase or        acetylcholinesterase with respect to motor neurons; or    -   (iv) decreased symptoms of neuron dysfunction in vivo.        Such effects may be measured by any method known in the art. In        preferred, non-limiting embodiments, increased survival of        neurons may be measured by the method set forth in Arakawa et        al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of        neurons may be detected by methods set forth in Pestronk et al.        (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev.        Neurosci. 4:17-42); increased production of neuron-associated        molecules may be measured by bioassay, enzymatic assay, antibody        binding, Northern blot assay, etc., depending on the molecule to        be measured; and motor neuron dysfunction may be measured by        assessing the physical manifestation of motor neuron disorder,        e.g., weakness, motor neuron conduction velocity, or functional        disability.

In a specific embodiments, motor neuron disorders that may be treatedaccording to the invention include but are not limited to disorders suchas infarction, infection, exposure to toxin, trauma, surgical damage,degenerative disease or malignancy that may affect motor neurons as wellas other components of the nervous system, as well as disorders thatselectively affect neurons such as amyotrophic lateral sclerosis, andincluding but not limited to progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

5.8.3. Tissue Repair and Regeneration

In another embodiment of the invention, a 10 Therapeutic of theinvention is used for promotion of tissue regeneration and repair,including but not limited to treatment of benign dysproliferativedisorders. Specific embodiments are directed to treatment of cirrhosisof the liver (a condition in which scarring has overtaken normal liverregeneration processes), treatment of keloid (hypertrophic scar)formation (disfiguring of the skin in which the scarring processinterferes with normal renewal), psoriasis (a common skin conditioncharacterized by excessive proliferation of the skin and delay in propercell fate determination), and baldness (a condition in which terminallydifferentiated hair follicles (a tissue rich in Notch) fail to functionproperly). In another embodiment, a Therapeutic of the invention is usedto treat degenerative or traumatic disorders of the sensory epitheliumof the inner ear.

5.9 Prophylactic Uses 5.9.1. Malignancies

The Therapeutics of the invention can be administered to preventprogression to a neoplastic or malignant state, including but notlimited to those disorders listed in Table 1. Such administration isindicated where the Therapeutic is shown in assays, as described supra,to have utility for treatment or prevention of such disorder. Suchprophylactic use is indicated in conditions known or suspected ofpreceding progression to neoplasia or cancer, in particular, wherenon-neoplastic cell growth consisting of hyperplasia, metaplasia, ormost particularly, dysplasia has occurred (for-review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

Alternatively or in addition to the presence of abnormal cell growthcharacterized as hyperplasia, metaplasia, or dysplasia, the presence ofone or more characteristics of a transformed phenotype, or of amalignant phenotype, displayed in vivo or displayed in vitro by a cellsample from a patient, can indicate the desirability ofprophylactic/therapeutic administration of a Therapeutic of theinvention. As mentioned supra, such characteristics of a transformedphenotype include morphology changes, looser substratum attachment, lossof contact inhibition, loss of anchorage dependence, protease release,increased sugar transport, decreased serum requirement, expression offetal antigens, disappearance of the 250,000 dalton cell surfaceprotein, etc. (see also id., at pp. 84-90 for characteristics associatedwith a transformed or malignant phenotype).

In a specific embodiment, leukoplakia, a benign-appearing hyperplasticor dysplastic lesion of the epithelium, or Bowen's disease, a carcinomain situ, are pre-neoplastic lesions indicative of the desirability ofprophylactic intervention.

In another embodiment, fibrocystic disease (cystic hyperplasia, mammarydysplasia, particularly adenosis (benign epithelial hyperplasia)) isindicative of the desirability of prophylactic intervention.

In other embodiments, a patient which exhibits one or more of thefollowing predisposing factors for malignancy is treated byadministration of an effective amount of a Therapeutic: a chromosomaltranslocation associated with a malignancy (e.g., the Philadelphiachromosome for chronic myelogenous leukemia, t(14;18) for follicularlymphoma, etc.), familial polyposis or Gardner's syndrome (possibleforerunners of colon cancer), benign monoclonal gammopathy (a possibleforerunner of multiple myeloma), and a first degree kinship with personshaving a cancer or precancerous disease showing a Mendelian (genetic)inheritance pattern (e.g., familial polyposis of the colon, Gardner'ssyndrome, hereditary exostosis, polyendocrine adenomatosis, medullarythyroid carcinoma with amyloid production and pheochromocytoma,Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen,retinoblastoma, carotid body tumor, cutaneous melanocarcinoma,intraocular melanocarcinoma, xeroderma pigmentosum, ataxiatelangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplasticanemia, and Bloom's syndrome; see Robbins and Angell, 1976, BasicPathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 112-113) etc.)

In another specific embodiment, an Antagonist Therapeutic of theinvention is administered to a human patient to prevent progression tobreast, colon, or cervical cancer.

5.9.2. Other Disorders

In other embodiments, a Therapeutic of the invention can be administeredto prevent a nervous system disorder described in Section 5.8.2, orother disorder (e.g., liver cirrhosis, psoriasis, keloids, baldness)described in Section 5.8.3.

5.10. Demonstration of Therapeutic or Prophylactic Utility

The Therapeutics of the invention can be tested in vivo for the desiredtherapeutic or prophylactic activity. For example, such compounds can betested in suitable animal model systems prior to testing in humans,including but not limited to rats, mice, chicken, cows, monkeys,rabbits, etc. For in vivo testing, prior to administration to humans,any animal model system known in the art may be used.

5.11. Antisense Regulation of Delta Expression

The present invention provides the therapeutic or prophylactic use ofnucleic acids of at least six nucleotides that are antisense to a geneor cDNA encoding Delta or a portion thereof. “Antisense” as used hereinrefers to a nucleic acid capable of hybridizing to a portion of a DeltaRNA (preferably mRNA) by virtue of some sequence complementarity. Suchantisense nucleic acids have utility as Antagonist Therapeutics of theinvention, and can be used in the treatment or prevention of disordersas described supra in Section 5.8 and its subsections.

The antisense nucleic acids of the invention can be oligonucleotidesthat are double-stranded or single-stranded, RNA or DNA or amodification or derivative thereof, which can be directly administeredto a cell, or which can be produced intracellularly by transcription ofexogenous, introduced sequences.

In a specific embodiment, the Delta antisense nucleic acids provided bythe instant invention can be used for the treatment of tumors or otherdisorders, the cells of which tumor type or disorder can be demonstrated(in vitro or in vivo) to express a Delta gene or a Notch gene. Suchdemonstration can be by detection of RNA or of protein.

The invention further provides pharmaceutical compositions comprising aneffective amount of the Delta antisense nucleic acids of the inventionin a pharmaceutically acceptable carrier, as described infra in Section5.12. Methods for treatment and prevention of disorders (such as thosedescribed in Sections 5.8 and 5.9) comprising administering thepharmaceutical compositions of the invention are also provided.

In another embodiment, the invention is directed to methods forinhibiting the expression of a Delta nucleic acid sequence in aprokaryotic or eukaryotic cell comprising providing the cell with aneffective amount of a composition comprising an antisense Delta nucleicacid of the invention.

Delta antisense nucleic acids and their uses are described in detailbelow.

5.11.1. Delta Antisense Nucleic Acids

The Delta antisense nucleic acids are of at least six nucleotides andare preferably oligonucleotides (ranging from 6 to about 50oligonucleotides). In specific aspects, the oligonucleotide is at least10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or atleast 200 nucleotides. The oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides, oragents facilitating transport across the cell membrane (see, e.g.,Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCTPublication No. WO 88/09810, published Dec. 15, 1988) or blood-brainbarrier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25,1988), hybridization-triggered cleavage agents (see, e.g., Krol et al.,1988, BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon,1988, Pharm. Res. 5:539-549).

In a preferred aspect of the invention, a Delta antisenseoligonucleotide is provided, preferably of single-stranded DNA. In amost preferred aspect, such an oligonucleotide comprises a sequenceantisense to the sequence encoding an SH3 binding domain or aNotch-binding domain of Delta, most preferably, of human Delta. Theoligonucleotide may be modified at any position on its structure withsubstituents generally known in the art.

The Delta antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide comprises at least onemodified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the oligonucleotide is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641).

The oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

In a specific embodiment, the Delta antisense oligonucleotide comprisescatalytic RNA, or a ribozyme (see, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). In another embodiment, the oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

In an alternative embodiment, the Delta antisense nucleic acid of theinvention is produced intracellularly by transcription from an exogenoussequence. For example, a vector can be introduced in vivo such that itis taken up by a cell, within which cell the vector or a portion thereofis transcribed, producing an antisense nucleic acid (RNA) of theinvention. Such a vector would contain a sequence encoding the Deltaantisense nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theDelta antisense RNA can be by any promoter known in the art to act inmammalian, preferably human, cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a Deltagene, preferably a human Delta gene. However, absolute complementarity,although preferred, is not required. A sequence “complementary to atleast a portion of an RNA,” as referred to herein, means a sequencehaving sufficient complementarity to be able to hybridize with the RNA,forming a stable duplex; in the case of double-stranded Delta antisensenucleic acids, a single strand of the duplex DNA may thus be tested, ortriplex formation may be assayed. The ability to hybridize will dependon both the degree of complementarity and the length of the antisensenucleic acid. Generally, the longer the hybridizing nucleic acid, themore base mismatches with a Delta RNA it may contain and still form astable duplex (or triplex, as the case may be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

5.11.2. Therapeutic Utility of Delta Antisense Nucleic Acids

The Delta antisense nucleic acids can be used to treat (or prevent)malignancies or other disorders, of a cell type which has been shown toexpress Delta or Notch. In specific embodiments, the malignancy iscervical, breast, or colon cancer, or squamous adenocarcinoma.Malignant, neoplastic, and pre-neoplastic cells which can be tested forsuch expression include but are not limited to those described supra inSections 5.8.1 and 5.9.1. In a preferred embodiment, a single-strandedDNA antisense Delta oligonucleotide is used.

Malignant (particularly, tumor) cell types which express Delta or NotchRNA can be identified by various methods known in the art. Such methodsinclude but are not limited to hybridization with a Delta orNotch-specific nucleic acid (e.g. by Northern hybridization, dot blothybridization, in situ hybridization), observing the ability of RNA fromthe cell type to be translated in vitro into Notch or Delta,immunoassay, etc. In a preferred aspect, primary tumor tissue from apatient can be assayed for Notch or Delta expression prior to treatment,e.g., by immunocytochemistry or in situ hybridization.

Pharmaceutical compositions of the invention (see Section 5.12),comprising an effective amount of a Delta antisense nucleic acid in apharmaceutically acceptable carrier, can be administered to a patienthaving a malignancy which is of a type that expresses Notch or Delta RNAor protein.

The amount of Delta antisense nucleic acid which will be effective inthe treatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. Where possible, it is desirable to determine theantisense cytotoxicity of the tumor type to be treated in vitro, andthen in useful animal model systems prior to testing and use in humans.

In a specific embodiment, pharmaceutical compositions comprising Deltaantisense nucleic acids are administered via liposomes, microparticles,or microcapsules. In various embodiments of the invention, it may beuseful to use such compositions to achieve sustained release of theDelta antisense nucleic acids. In a specific embodiment, it may bedesirable to utilize liposomes targeted via antibodies to specificidentifiable tumor antigens (Leonetti et al., 1990, Proc. Natl. Acad.Sci. U.S.A. 87:2448-2451; Renneisen et al., 1990, J. Biol. Chem.265:16337-16342).

5.12. Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a Therapeutic ofthe invention. In a preferred aspect, the Therapeutic is substantiallypurified. The subject is preferably an animal, including but not limitedto animals such as cows, pigs, chickens, etc., and is preferably amammal, and most preferably human.

Various delivery systems are known and can be used to administer aTherapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, expression by recombinant cells,receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as partof a retroviral or other vector, etc. Methods of introduction includebut are not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompounds may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by infection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

In another embodiment, the Therapeutic can be delivered in a vesicle, inparticular a liposome (see Langer, Science 249:1527-1533 (1990); Treatet al., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the Therapeutic can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled rug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the Therapeutic is a nucleic acidencoding a protein Therapeutic, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid Therapeuticcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination.

In specific embodiments directed to treatment or prevention ofparticular disorders, preferably the following forms of administrationare used:

Preferred Forms of Disorder Administration Cervical cancer TopicalGastrointestinal cancer Oral; intravenous Lung cancer Inhaled;intravenous Leukemia Intravenous; extracorporeal Metastatic carcinomasIntravenous; oral Brain cancer Targeted; intravenous; intrathecal Livercirrhosis Oral; intravenous Psoriasis Topical Keloids Topical BaldnessTopical Spinal cord injury Targeted; intravenous; intrathecalParkinson's disease Targeted; intravenous; intrathecal Motor neurondisease Targeted; intravenous; intrathecal Alzheimer's disease Targeted;intravenous; intrathecal

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the Therapeutic, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5%to 10% by weight; oral formulations preferably contain 10% to 95% activeingredient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

5.13. Diagnostic Utility

Delta proteins, analogues, derivatives, and subsequences thereof, Deltanucleic acids (and sequences complementary thereto), anti-Deltaantibodies, have uses in diagnostics. Such molecules can be used inassays, such as immunoassays, to detect, prognose, diagnose, or monitorvarious conditions, diseases, and disorders affecting Delta expression,or monitor the treatment thereof. In particular, such an immunoassay iscarried out by a method comprising contacting a sample derived from apatient with an anti-Delta antibody under conditions such thatimmunospecific binding can occur, and detecting or measuring the amountof any immunospecific binding by the antibody. In a specific aspect,such binding of antibody, in tissue sections, preferably in conjunctionwith binding of anti-Notch antibody can be used to detect aberrant Notchand/or Delta localization or aberrant levels of Notch-Deltacolocalization in a disease state. In a specific embodiment, antibody toDelta can be used to assay in a patient tissue or serum sample for thepresence of Delta where an aberrant level of Delta is an indication of adiseased condition. Aberrant levels of Delta binding ability in anendogenous Notch protein, or aberrant levels of binding ability to Notch(or other Delta ligand) in an endogenous Delta protein may be indicativeof a disorder of cell fate (e.g., cancer, etc.) By “aberrant levels,” ismeant increased or decreased levels relative to that present, or astandard level representing that present, in an analogous sample from aportion of the body or from a subject not having the disorder.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew.

Delta genes and related nucleic acid sequences and subsequences,including complementary sequences, and other toporythmic gene sequences,can also be used in hybridization assays. Delta nucleic acid sequences,or subsequences thereof comprising about at. least 8 nucleotides, can beused as hybridization probes. Hybridization assays can be used todetect, prognose, diagnose, or monitor conditions, disorders, or diseasestates associated with aberrant changes in Delta expression and/oractivity as described supra. In particular, such a hybridization assayis carried out by a method comprising contacting a sample containingnucleic acid with a nucleic acid probe capable of hybridizing to DeltaDNA or RNA, under conditions such that hybridization can occur, anddetecting or measuring any resulting hybridization.

Additionally, since Delta binds to Notch, Delta or a binding portionthereof can be used to assay for the presence and/or amounts of Notch ina sample, e.g., in screening for malignancies which exhibit increasedNotch expression such as colon and cervical cancers.

6. A Delta Homolog in the Chick is Expressed in Prospective Neurons

As described herein, we have isolated and characterized a chick Deltahomologue, C-Delta-1. We show that C-Delta-1 is expressed in prospectiveneurons during neurogenesis, as new cells are being born and their fatesdecided. Our data in the chick, suggest that both the Delta/Notchsignalling mechanism and its role in neurogenesis have been conserved invertebrates.

6.1. Cloning of C-Delta-1

We identified a chick Delta homologue, C-Delta-1, using the polymerasechain reaction (PCR) and degenerate oligonucleotide primers (FIGS.1A1-1A3, 1A3, 1B1-1B2 and 2, SEQ ID NOS:1, 5 2, 3 and 4). C-Delta-1 wascloned by PCR using the degenerate oligonucleotide primersTTCGGITT(C/T)ACITGGCCIGGIAC (SEQ ID NO:81) and TCIATGCAIGTICCICC(A/G)TT(SEQ ID NO:82) which correspond to the fly Delta protein sequencesFGFTWPGT (SEQ ID NO:83) and NGGTCID (SEQ ID NO:84), respectively (Vässinet al., 1987, EMBO J. 6:3431-3440; Kopczynski et al., 1988, Genes Dev.2:1723-1735). The initial reaction used Song of first-strandoligo-d(T)-primed cDNA from stage 4-6 embryos, 1 μg of each primer, 0.2mM dNTPs, 2U. of Taq polymerase, in 50 μl of the supplied buffer(Perkin-Elmer). 40 cycles of amplification were performed at 94° C./30sec; 50° C./2 min; 72° C./2 min. Amplified DNA fragments were separatedon an agarose gel, cloned in Bluescript pKS-(Stratagene) and sequenced.Two Delta homologs were identified, one of which (C-Delta-1) isexpressed in the nervous system. Of the homolog that is expressed in thenervous system, two variants were identified that differ at thecarboxy-terminal end of the encoded protein due to an alternativesplicing event at the 3′ end of the C-Delta-1 gene. One encoded proteinhas 12 extra amino acids at the carboxy-terminal end, relative to theother encoded protein. The sequence of the shorter encoded variant isset forth in SEQ ID NO:2. The longer variant encoded by SEQ ID NO:3 andidentified by the amino acid sequence of SEQ ID NO:4, consists of theamino acid sequence of SEQ ID NO:2 plus twelve additional amino acids atthe 3′ end (SIPPGSRTSLGV)(SEQ ID NO:85). The longer variant was used inthe experiments described below. When tested for biological activity byinjection of RNA into Xenopus oocytes, each of the variants had the samebiological activity.

DNA fragments corresponding to C-Delta-1 were used to screen a stage 17spinal cord cDNA library and several full-length clones were obtainedand sequenced. We amplified DNA fragments from chick C-Notch-1 gene bysimilar methods (data not shown); partial sequence data and pattern ofexpression indicate close similarity to the rodent Notch-1 gene(Weinmaster et al., 1991, Development 113:199-205; Weinmaster et al.,1992, Development 116:931-941; Lardelli & Lendahl, 1993, Exp. Cell Res.204:364-372). Sequences were analyzed using the Wisconsin GCG set ofprograms. The GenBank Accession number for the Chick Delta-1 mRNA isU26590. The DNA sequence of C-Delta-1 corresponds to a protein of 722amino acids, structurally homologous to Drosophila Delta (FIGS. 3A-3B,4) and clearly distinct from vertebrate homologs of the Delta-relatedSerrate protein, which we have also cloned (data not shown). C-Delta-1contains a putative transmembrane domain, a signal sequence IS and 8EGF-like repeats in its extracellular region (one repeat less thanDrosophila Delta). The amino-terminal domain of C-Delta-1 is closelyrelated to a similar domain in the fly Delta protein, described asnecessary and sufficient for in vitro binding to Notch (Muskavitch,1994, Dev. Biol. 166:415-430). This conserved region includes theso-called DSL motif (FIG. 4) (Henderson et al., 1994, Development120:2913-2924; Tax et al., 1994, Nature 368:150-154), shared by allknown members of the family of presumed ligands of Notch-like proteins(Delta and Serrate in Drosophila; Lag-2 and Apx-1 in Caenorhabditiselegans) (Henderson et al., 1994, Development 120:2913-2924; Tax et al.,1994, Nature 368:150-154; Fleming et al., 1990, Genes Dev. 4:2188-2201;Thomas et al., 1991, Development 111:749-761; Mello et al., 1994, Cell77:95-106). A second cysteine-rich N-terminal region is conservedbetween the fly and chick proteins, but absent from the related C.elegans proteins (FIG. 4). The Xenopus Delta-1 homologue, X-Delta-1which encodes a protein that is 81% identical to C-Delta-1 and shows allthe above structural motifs (FIG. 3A-3B), has also been cloned. Thestructural conservation between the chick and fly Delta proteins,including domains identified as critical for Notch binding (Muskavitch,1994, Dev. Biol. 166:415-430), suggeststhat C-Delta-1 functions as aligand for a chick Notch protein, and that a Delta/Notch-mediatedmechanism of lateral inhibition might operate in the chick.

6.2. C-Delta-1 and C-Notch-1 Expression Correlates with Onset ofNeurogenesis

During Drosophila neurogenesis, Delta is transiently expressed in neuralprecursors, inhibiting neighboring Notch-expressing cells from alsobecoming neural (Haenlin et al., 1990, Development 110:905-914; Kooh etal., 1993, Development 117:493-507). If C-Delta-i acts similarly duringchick neurogenesis, it should also be transiently expressed in neuronalprecursor cells, while these are becoming determined. An analysis ofC-Delta-1 expression in the developing CNS indicates that this is indeedthe case.

In summary, wholemount in situ hybridization was performed. Formaldehydefixed embryos were treated with protease and refixed with 4%formaldehyde/0.1% glutaraldehyde. Hybridization with DIG-labelled RNAprobes was performed under stringent conditions (1.3×SSC, 50% formamide,65° C., pH5) in a buffer containing 0.2% Tween-20 and 0.5% CHAPS. Washedembryos were treated with Boehringer Blocking Reagent and incubatedovernight in alkaline phosphatase-coupled anti-DIG antibody. Afterextensive washes, embryos were stained from 30 min to overnight. Theembryo in FIG. 5 e was wax-sectioned after hybridization.

C-Delta-1 expression in the neural plate is first detected at stage 6-7(31 h, 0/1 somite), in scattered cells just anterior to the presomiticmesoderm (FIG. 5 b, 5 c). This region gives rise to the mid/posteriorhindbrain, where the earliest differentiated CNS neurons are firstdetected by a neurofilament antibody at stage 9 (31 h, 7-9 somites)(Sechrist & Bronner-Fraser, 1991, Neuron 7:947-963), 6 h after theinitial C-Delta-1 expression (Table 2).

TABLE 2 Hamburger-Hamilton Stage (nominal age in h; somite nos.) InitialNeural tube End final C-Delta-1 Initial NF domains S-phase expressionexpression Mid/posterior 4 6  9 Hindbrain (19 h; 0) (24 h; 0) (31 h;7-9) Spinal cord, 6 8 10 somites 5-8 (24 h; 0) (28 h; 4-6) (36 h; 10-12)Forebrain/ 7 8 10 Midbrain (25 h; 1-3) (28 h; 4-6) (36 h; 10-12) Spinalcord, 8 9 11 somites 9-12 (28 h; 4-6) (31 h; 7-9) (43 h; 13-15)

As neurogenesis proceeds, expression of C-Delta-1 continues toforeshadow the spatio-temporal pattern of neuronal differentiation(Table 2), spreading posteriorly along the spinal cord and anteriorlyinto the midbrain and forebrain (FIG. 5 d, 5 e). For example, the mostposterior expressing cells in the stage 8 spinal cord are at the levelof the prospective 6th somite, 6-8 h before the first neurons at thatlevel express neurofilament antigen (Sechrist & Bronner-Fraser, 1991,Neuron 7:947-963) (Table 2). Table 2 shows that the appearance ofC-Delta-1 expression closely follows the withdrawal of the firstneuronal precursors from the division cycle and precedes the appearanceof neurofilament (NF) antigen in the resultant differentiating neurons.Mid-hindbrain comprises rhombomeres 4-6, the level of the oticprimordium; posterior hindbrain includes rhombomeres 7 and 8, andsomites 1-4. Data for the timing of withdrawal from cell-division andfor neurofilament expression are taken from Sechrist et al., 1991,Neuron 7:947-963. In all cases, C-Delta-1 is expressed in scatteredcells within domains of uniform C-Notch-1 expression (FIG. 5 a).

6.3. Localization and Time-Course Expression of C-Delta-1

The localization and time-course of C-Delta-1 expression indicate thatthe gene is switched on at an early step in neurogenesis, and that thecells expressing C-Delta-1 are prospective neurons that have not yetbegun to display differentiation markers. To test this hypothesis, wemade use of the observations of Sechrist and Bronner-Fraser (Sechrist &Bronner-Fraser, 1991, Neuron 7:947-963) that prospective neurons are theonly non-cycling cells in the early neural tube. They finish their finalS phase 11-15 h before expressing neurofilament antigen (Table 2) andtheir nuclei, after completing a last mitosis, adopt a characteristiclocation near the basal surface of the neuroepithelium, where all theother cell nuclei are in S-phase (Sechrist & Bronner-Fraser, 1991,Neuron 7:947-963; Martin & Langman, 1965, J. Embryol. Exp. Morphol.14:23-35) (FIG. 6 a). We labelled stage 7-9 embryos withbromodeoxyuridine (BrdU), and double-stained for BrdU incorporation andC-Delta-1 expression. 95% of the C-Delta-1-expressing cells wereunlabelled, with their nuclei predominantly located near the basalsurface, where most other nuclei were BrdU-labelled (FIG. 6 b, 6 c). 75μl 0.1 mM BrdU in PBS was dropped onto stage 7-9 embryos which wereincubated at 38° C. for 2-4 h before fixation for in situ hybridization.15 μm cryostat sections were hybridized with DIG-labelled RNA probes,essentially according to the method of Strahle et al. (Strahle et al.,1994, Trends In Genet. Sci. 10:75-76). After staining, slides werewashed in PBS, and processed for BrdU immunodetection (Biffo et al.,1992, Histochem. Cytochem. 40:535-540). Anti-BrdU (1:1000; Sigma) wasdetected using FITC-coupled goat anti-mouse secondary antibody (Cappel).C-Delta-1 expression was examined by DIC microscopy, and BrdU-labellingby conventional and confocal fluorescence microscopy. These resultsimply that C-Delta-1 is expressed in cells that have withdrawn from thecell cycle and must indeed be prospective neurons. The fewBrdU⁺/C-Delta-1+ cells have their nuclei outside the basal zone; thesemay be cells that finished their final S-phase soon after exposure toBrdU, moved apically to complete their final mitosis, and switched onC-Delta-1 expression. C-Delta-1 is also expressed in the later neuraltube and peripheral nervous system. Again, the timing of expression andthe location of the expressing cells imply that they are neuronalprecursors that have not yet begun to differentiate (data not shown).Thus, C-Delta-1 expression appears to be the earliest known marker forprospective neurons.

In addition, the transcription pattern of both C-Delta-1 and C-Serrate-1overlap that of C-Notch-1 in many regions of the embryo (data not shown)which suggest that C-Notch-1, like Notch in Drosophila, is a receptorfor both proteins. In particular, all three genes are expressed in theneurogenic region of the developing central nervous system, and here astriking relationship is seen: the expression of both C-Serrate-1 andC-Delta-1 is confined to the domain of C-Notch-1 expression; but withinthis domain, the regions of C-Serrate-1 and C-Delta-1 are preciselycomplementary. The overlapping expression patterns suggest conservationof their functional relationship with Notch and imply that developmentof the chick and in particular the central nervous system involves theconcerted interaction of C-Notch-1 with different ligands at differentlocations.

6.4. Discussion

The Xenopus homolog of C-Delta-1 has been cloned in a similar manner. Inbrief, a PCR fragment of X-Delta-1 was isolated and sequenced. Thisfragment was then used to identify the full length clone of X-Delta-1.The X-Delta-1 expression pattern was studied. It was shown thatX-Delta-1 is expressed in scattered cells in the domain of the neuralplate where primary neuronal precursors are being generated, suggestingthat the cells expressing X-Delta-1 are the prospective primary neurons.In addition, X-Delta-1 is also expressed at other sites and times ofneurogenesis, including the anterior neural plate and neurogenicplacodes and later stages of neural tube development when secondaryneurons are generated. Ectopic X-Delta-1 activity inhibited productionof primary neurons; interference with endogenous X-Delta-1 activityresulted in overproduction of primary neurons. These results show thatX-Delta-1 mediates lateral inhibition delivered by prospective neuronsto adjacent cells. It was shown that ectopic expression of X-Delta-1 inXenopus eggs suppresses primary neurogenesis, and that ectopicexpression of a truncated X-Delta-1 protein which retains only two aminoacids of the cytoplasmic domain interferes with endogenous signallingand leads to extra cells developing as neuronal precursors. (Chitnis etal., Nature (in press). Preliminary evidence indicates that C-Delta-1has a similar inhibitory action when expressed in Xenopus embryos (datanot shown). We propose that C-Delta-1, like its Drosophila and Xenopuscounterparts, mediates lateral inhibition throughout neurogenesis torestrict the proportion of cells that, at any time, become committed toa neural fate. C-Delta-1 is generally expressed during neurogenesis inmany other sites, in both the CNS and PNS, and, for example, thedeveloping ear. It has been shown in the CNS that C-Notch is expressedin the ventricular zone of the E5 chick hindbrain, in dividing cellsadjacent to the lumen of the neural tube. C-Delta-1 is expressed in theadjacent layer of cells, which have stopped dividing and are becomingcommitted as neuronal precursor cells. Thus, Delta/Notch signallingcould act here, as in other neural tissues, to maintain a population ofuncommitted cycling neuronal stem cells.

7. Isolation and Characterization of a Mouse Delta Homolog

A mouse Delta homolog, termed M-Delta-1, was isolated as follows:

Mouse Delta-1 gene

Tissue Origin: 8.5 and 9.5-day mouse embryonic RNA Isolation Method:

-   -   a) random primed CDNA against above RNA    -   b) PCR of above CDNA using

PCR primer 1: GGITTCACITGGCCIGGIACNTT (SEQ ID NO:86) [encoding GFTWPGTF(SEQ ID NO:94), a region which is specific for Delta-, not Serrate-likeproteins]

PCR primer 2: GTICCICC(G/A)TT(C/T)TT(G/A)CAIGG(G/A)TT (SEQ ID NO:87)[encoding NPCKNGGT (SEQ ID NO:88), a sequence present in many of theEGF-like repeats]

Amplification conditions: 50 ng CDNA, 1 μg each primer, 0.2 mM dNTP's,1.8 U Taq (Perkin-Elmer) in 50 μl of supplied buffer. 40 cycles of: 94°C./30 sec, 45° C./2 min, 72° C./1 min extended by 2 sec each cycle.

The amplified fragment was an approximately 650 base pair fragment whichwas partially sequenced to determine its relationship to C-Delta-1.

-   -   c) a mouse 11.5 day cDNA library (Clontech) was screened. Of        several positive clones, one (pMDL2; insert size approximately 4        kb) included the complete protein-coding region whose DNA        sequence was completely determined.

FIGS. 7A-7B (SEQ ID NO:11) show the nucleotide sequence of the isolatedclone containing M-Delta-1 DNA.

FIG. 8 (SEQ ID NO:12) shows the predicted amino acid sequence ofM-Delta-1.

FIGS. 9A-9B shows an amino acid alignment of the predicted amino acidsequences for M-Delta-1 and C-Delta-1. Identical amino acids are boxedshowing the extensive sequence homology. The consensus sequence is shownbelow (SEQ ID NO:13).

Expression pattern: The expression pattern was determined to beessentially the same as that observed for C-Delta-1, in particular, inthe presomitic mesoderm, central nervous system, peripheral nervoussystem, and kidney.

8. Isolation and Characterization of a Human Delta Homolog

A human Delta-1 homolog, termed H-Delta-1 (HD1), was isolated asfollows:

A human genomic library with inserts ranging in size from 100-150 kb wasprobed with an EcoRI fragment of the mouse Delta-1 (M-Delta-1) gene.From the library a genomic human PAC clone was isolated which hybridizedto the EcoRI fragment. Next, two degenerate oligonucleotides were usedto amplify by PCR a fragment of the genomic human PAC clone. Thedegenerate oligos were:

5′ ACIATGAA(C/T)AA(C/T)CTIGCIAA(C/T)TG (SEQ ID NO:89) [encoding TMNNLANC(SEQ ID NO:90)] and

3′ AC(A/G)TAIACIGA(C/T)TG(A/G)TA(C/T)TTIGT (SEQ ID NO:91) [encodingTKYQSVYV (SEQ ID NO:92] or

3′ GC(A/G/T)ATIAC(A/G)CA(C/T)TC(A/G)TC(C/T)TT(C/T)TC (SEQ ID NO:93)[encoding EKDECVIA (SEQ ID NO:25].

On the basis of the cDNA sequences for chicken and mouse Delta-1, it wasexpected that fragments of approximately 354 and 387 base pairs would beisolated, using the 5′ and the two different 3′ oligos, respectively. Infact, however, two single isolates of 525 base pairs and another thatwas 30 base pairs smaller, as expected, were obtained. The largerisolate was sequenced by dideoxy sequencing. The nucleotide sequence isshown in FIG. 10A-10B (SEQ ID NO:14). Also shown in FIG. 10A-10B are thepredicted amino acid sequences of the amplified DNA fragment (SEQ IDNOS:15-22) for the three different readings frames. Due to sequencingerrors, the full uninterrupted sequence between both primers was notidentified. As a consequence, one cannot predict the amino acid sequencedirectly from the DNA sequence obtained. However, FIG. 11 shows theamino acid sequence homology between human Delta-1 (top line) (SEQ IDNO:23) and chick Delta-1 (bottom line) as determined by eye. Because ofthe sequencing errors, the homology was obtained by switching amongstthe three different reading frames to identify the homologous regions.

Using the larger isolate (SEQ ID NO:14) as probe, a human fetal brainplasmid library (Clontech) was screened in an attempt to isolatefull-length H-Delta-1 (HD1) genes. This yielded four positive plaques.Two of these positives (HD13 and HD124) survived rescreening and reactedpositively with a large human genomic fragment on a Southern Blot. Thesepositive clones were subcloned by digesting with EcoRI and ligating thefragments into a Bluescript KS-vector. The nucleotide sequences of theinserts were obtained by dideoxy sequencing using T3 and T7 primers. Theresults showed that HD124 was homologous to chicken Delta-1 at bothends; however, one end of HD13 showed no homology. Restrictiondigestions with a panel of enzymes showed very similar patterns betweenthe two clones, each of which had an insert of about 2 kb, but withdifferences at the 3′ end of HD13.

HD13 and HD124 were cut with BstXI, XbaI, HindIII and XhoI and therestriction fragments were inserted into Bluescript KS⁻, and thensequenced as described above to obtain internal sequence. The sequencethat was obtained represents the 3′ about 2000 bases of HD1, extendinginto the 3′ non-coding region. HD13 is contained within HD124; however,the added sequence at the 5′ end of HD13 is likely due to a cloningartifact.

Since the sequence thus obtained did not contain the 5′ end of HD1,HD124 was used as a probe for subsequent hybridizations in a T celllibrary and in another fetal brain library (Lambda-Zap, Stratagene). Ascreen of the T cell library resulted in no positives. However,screening the Lambda-Zap library resulted in two positive clones, HDl13and HD118. These clones were inserted into a Bluescript KS-vector usingEcoRI as described above. The inserts were digested with a panel ofrestriction enzymes for comparison with HD13 and HD124, and the 5′ and3′ ends were sequenced using T3 and T7 primers. HD113 was determined tobe only a small piece of cDNA that when sequenced showed no homology toany known Delta. However, HD118 was 3 kb in length, and included theentire sequence of HD124 with additional 5′ sequences. A set of cloneswere isolated using nested deletions from HD118; these clones were thensubjected to dideoxy sequencing using an automated sequencer. FIG.12A1-12A3 presents the partial nucleotide contig sequence (SEQ ID NO:26)of human Delta obtained from clone HD118. Due to sequencing errors, thefull uninterrupted nucleotide sequence of human Delta was notdetermined. FIG. 12B1-12B6 show the partial nucleotide contig sequence(SEQ ID NO:26) of human Delta (top line), with the predicted amino acidsequence in three different reading frames presented below, the secondline being reading frame 1 (SEQ ID NO:27-42), the third line beingreading frame 2 (SEQ ID NO:43-47), and the fourth line being readingframe 3 (SEQ ID NO:48-64).

Sequence homology was determined by eye using the mouse Delta-1 aminoacid sequence. The sequences with the greatest degree of homology to themouse amino acid sequence are boxed in FIG. 12B1-12B6, and represent thepredicted amino acid sequence of human Delta-1. The composite resultingamino acid sequence is shown in FIG. 14A-14B. (In FIGS. 14A-14B, thevarious uninterrupted portions of the human Delta sequence are assignedrespectively, SEQ ID NOS:65-80.) Note that due to sequencing errors, thereading frame with the greatest homology is not the same throughout thesequence and shifts at positions where there are errors in the sequence.

Further, the homology determined by eye to chicken and mouse Deltaindicates that the amino acid sequence deduced from the determined humanDelta nucleotide sequence contains all but about the N-terminal 100-150amino acids of human Delta-1.

FIG. 13A-13G present the nucleotide sequence of mouse Delta-1 (top line,SEQ ID NO:4) and the contig nucleotide sequence of human Delta-1 asdepicted in FIGS. 12A1-12A3 and 12B1-12B6 (second line, SEQ ID NO:26)and the nucleotide consensus sequence between mouse and human Delta(third line, SEQ ID NO:24).

Using probes containing the human Delta 5′ nucleotide sequencespresented in FIGS. 12A1-12A3, cDNA libraries are probed to isolate the5′ end of the human Delta gene. Primary positive clones are obtained andthen confirmed as secondary positives. The secondary positives arepurified and grown further. The DNA is then isolated and subcloned forsequencing.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method of treating a disease or disorder in asubject comprising administering to a subject in which such treatment isdesired a therapeutically effective amount of a fragment of a nativehuman Delta protein, which native human Delta protein comprises 20continuous amino acids of SEQ ID NO:23, and which fragment (i) is insoluble form, (ii) comprises the DSL domain of the native human Deltaprotein, and (iii) is able to bind to a Notch protein and antagonizeNotch function; which disease or disorder is a malignancy characterizedby increased Notch activity or increased expression of a Notch proteinor of a Notch derivative capable of binding a Delta protein and of beingbound by an antibody to a Notch protein, relative to said Notch activityor expression in an analogous non-malignant sample.
 2. The methodaccording to claim 1 in which the fragment is not larger than 100 aminoacids.
 3. The method according to claim 1 in which the fragment is notlarger than 200 amino acids.
 4. The method according to claim 1 in whichthe fragment lacks the transmembrane and intracellular domains of thehuman Delta protein.
 5. The method according to claim 1 in which thedisease or disorder is selected from the group consisting of cervicalcancer, breast cancer, colon cancer, melanoma, and lung cancer.
 6. Themethod according to claim 1 in which the subject s a human.
 7. Themethod according to claim 1 in which the fragment lacks theintracellular domain of the human Delta protein.